Subsyndromal symptomatic depression (SSD) is a subtype of subthreshold depressive and also lead to significant psychosocial functional impairment as same as major depressive disorder (MDD). Several studies have suggested that SSD is a transitory phenomena in the depression spectrum and is thus considered a subtype of depression. However, the pathophysioloy of depression remain largely obscure and studies on SSD are limited. The present study compared the expression profile and made the classification with the leukocytes by using whole-genome cRNA microarrays among drug-free first-episode subjects with SSD, MDD, and matched controls (8 subjects in each group). Support vector machines (SVMs) were utilized for training and testing on candidate signature expression profiles from signature selection step. Firstly, we identified 63 differentially expressed SSD signatures in contrast to control (P< = 5.0E-4) and 30 differentially expressed MDD signatures in contrast to control, respectively. Then, 123 gene signatures were identified with significantly differential expression level between SSD and MDD. Secondly, in order to conduct priority selection for biomarkers for SSD and MDD together, we selected top gene signatures from each group of pair-wise comparison results, and merged the signatures together to generate better profiles used for clearly classify SSD and MDD sets in the same time. In details, we tried different combination of signatures from the three pair-wise compartmental results and finally determined 48 gene expression signatures with 100% accuracy. Our finding suggested that SSD and MDD did not exhibit the same expressed genome signature with peripheral blood leukocyte, and blood cell–derived RNA of these 48 gene models may have significant value for performing diagnostic functions and classifying SSD, MDD, and healthy controls. Depression affects about 10% of the population at some point in their life and is the leading cause of disability across the world 12. Furthermore, several follow-up studies have suggested that SSD is a transitory phenomena in the depression spectrum and is thus considered a subtype of depression Although the pathophysioloy of depression spectrum remain largely obscure, it has been reported that patients with SSD and MDD have similar family history, and their first-degree relatives have a high risk of comorbidity of depression and alcohol dependence, which implies that these two disorders could share genetic basesWith the sequence of the human genome being publicly available since February 2001, an array of novel research tools, such as gene expression microarray, have become available that may yield unbiased, hypothesis-free insight into the pathophysiologic underpinnings of this disorder Disease development is a systematic and dynamic processes influenced by environment factors and genetic factors, together. Computational and systems biology have greatly facilitate the disease studies from transcriptomes by using microarray technology With gene features made on microarray accumulated by technology developing, many psychiatric disorder studies are also applied by the high-throughput technology with bioinformatics analysis. Tsuang et.,al have assessed the validity of blood-based gene expression profiles for the classification of schizophrenia and bipolar disorder Thus, in order to develop the potential peripheral blood lymphocytes gene expression signature models which can classify MDD, SSD, and healthy controls, whole-genome cRNA microarray analysis of lymphocytes were performed in this study.For SSD gene expression signatures, we detected 1,456 differential expressed genes between SSD and healthy controls, in which 753 genes are up regulated and 703 genes are down regulated (adjusted p<0.01), which enriched in 47 pathways (P<0.01). Most of genes involved in several functional related to signaling pathways, including neuroactive ligand receptor interaction, JAK and STAT signaling pathway, G protein signaling, calcium signaling pathway, insulin signaling pathway, GNRH signaling pathway, Wnt signaling pathway and MAPK signaling pathway etc. Cellular communication and cell structure organization were also important in SSD process, such as apoptosis, cell adhesion molecules, tight junction, focal adhesion. The DEG also act in several biosynthesis and metabolism pathways, like oxidative phsphorylation, metabolism of xenobiotic by cytochrome P450, purine metabolism, glycerlipid metabolism, glycan structures biosynthesis, glycerolipid metabolism, starch and sucrose metabolism. We also found that SSD signatures participate in immunity process, antigen processing, leukocyte transendothelial migration, natural killer cell mediated cytototoxicity and cytokine-cytokine receptor interaction . GO analBased on pre-processed microarray profile, we identified 149 differential genes between MDD patients and controls with 95 upregulated and 54 down-regulated (adjusted P<0.01), 20 of which were identified between SSD and control. These differential genes enriched in 53 pathways, 2 of which also were identified in SSD. Signaling pathways active in MDD include activation of ATR in response to replication stress, NRIF signals cell death from the nucleus, fas signaling pathway, p53-Independent G1/S DNA damage checkpoint and Nicotinamide salvaging, and EGF signaling pathway etc. We noticed that many MDD signatures involves several immunity process, such as T cell receptor signaling pathway and JNK signaling in the CD4+ TCR pathway, IL2-mediated signaling events, IL1 signaling and IL6-mediated signaling events and Calcium signaling in the CD4+ TCR pathway and TCR signaling in CD4+ T cells. In MDD subjects, more biosynthesis and metabolism pathway are identified, involveing Vitamin B5 (pantothenate) metabolism, coenzyme A biosynthesis and metabolism of water-soluble vitamins and cofactors. Comparing with former results of SSD signatures, we found that several pathways were shared in MDD and SSD process, including cell cycle controls and Cell Cycle Checkpoints, like G2/M Checkpoints and Wnt signaling. We noticed that MDD-specific functions or pathways compared with SSD were activation of ATR pathway in response to replication stress, NRIF signals cell death from the nucleus, fas signaling pathway, immunity pathway about IL2 signaling events mediated by PI3K and IL1 singaling events. Meanwhile, SSD-specific pathways contain cytokine-cytokine receptor interaction, GPCRDB class A rhodopsin like, MAPK signaling pathway, neuroactive ligand receptor interaction, calcium signaling pathway, breast cancer estrogen signaling pathway, purine metabolism, insulin signaling pathway, cell adhesion molecules and Toll like receptor signaling pathway. AlternaIn order to filter out most of false positives and select most potential biomarkers, we applied strict threshold on the same pair-wise comparisons among SSD, MDD and controls, and identified 63 differentially expressed SSD signatures in contrast to controls (adjusted P< = 1.0E-4) and 30 differentially expressed MDD signatures in contrast to controls (adjusted P< = 5.0E-4), respectively. Then, 123 gene signatures were identified with significantly differential expression level between SSD and MDD (adjusted P< = 1.0E-4). Unsupervised hierarchal clustering analysis by using Euclidean distance and complete linkage clustering method was conducted on three more potential groups of DEGs. The results showed clearly that genes differentially expressed in the peripheral blood lymphocytes were capable of differentiating MDD group, SSD group and healthy controls, separately .To evaluate the predictive performances of SSD and MDD signatures respectively, we utilized SVM with Linear Kernel to build disorder models. For 63 SSD signatures (adjusted P< = 1.0E-4), total instances  were correctly classified. Similarly, for 30 MDD signatures (adjusted P< = 5.0E-4), and 123 DEGs signatures (adjusted P< = 1.0E-4) can also clearly classified 8 MDD and 8 SSD instances.For SSD gene signatures, we detected 1,456 differential expressed genes between SSD and control; For MDD gene signatures, we identified 149 differential genes between MDD patients and controls d. Among these genes, there are only 20 different genes between SSD and MDD.Furthermore, in order to conduct priority selection for biomarkers for SSD and MDD together, we selected top gene signatures from each group of pair-wise comparison results, and merged the signatures together to generate better profiles used for clearly classify SSD and MDD states in the same time. In details, we tried different combination of signatures from top ranked signatures in the three pair-wise compartmental results and finally determined 48 gene expression signatures . To mainWhen 54 probesets (48 genes) were chosen as biomarkers, we obtained the best predictive performances with 100% accuracy and 100% TPR . Leave-one-off validation refers to that we used n-1 sample to train model and used another sample to test the model. Total 24 MDD, SSD and control samples were separated into train and test profiles in 24 times and train/test the data using leave-one-off method. Finally, we collected the predictions for each sample, and obtained 100% predictive performance .Then, we interrogated the pathways and biological functions about these mostly potential biomarkers for MDD and SSD, together. Pathway analysis demonstrates that the potential 48 gene biomarkers involved in insulin signaling pathway, signaling by NGF, ErbB signaling pathway, neurotrophin signaling pathway, cell surface interactions at the vascular wall, NRAGE signals death through JNK, Rho GTPase cycle, and G alpha signaling pathway  . Also, GIn order to investigate how the signatures for classifying three groups are regulated, we analyzed the cis-regulaotry elements co-occurring on the promoters of these genes. In details, STAT1 and STAT2 factor' binding motifs were detected on five MDD signatures' promoters . In addition, we identified SRF binding motif on three gene promoters . For SSD signatures, we identified transcriptional factor SREBP1 was correlated with eleven SSD signature genes . Most of these transcriptional factors are related with depression disorders as discussed below.To our knowledge, this is the first study to compare the expression profile and make the classification with the leukocytes by using whole-genome cRNA microarrays among patients with SSD, major depressive disorder (MDD) and controls. We found that SSD and MDD had different blood-based gene expression signature, and the differential expressed genes of SSD were about 10 times of MDD, but there are only 20 overlapping differential expressed genes between SSD and MDD. Pathway analysis for SSD gene signatures showed that differential expressed genes enriched in 47 pathways, and most pathways were involved in regulation of DNA replication, IL2 signaling events mediated by STAT5, and Wnt signaling pathway, etc. For MDD gene signatures, the results of pathway analysis suggested that differential expressed genes enriched in 53 pathways, 2 of which also were identified in SSD, including MAPK signaling pathway and Wnt signaling pathway. Although the relationship between SSD and MDD is unclear, previous follow-up studies have showed that SSD was a subtype of depression and a transitory phenomenon in depression spectrum with a high likelihood of transition to MDD Genetic manipulation of the MAPK pathway, one of the neurotrophin signaling pathways, has received much attention, which postulated that the dysfunction of this pathway played a key role in the pathophysiology of mood disorders Wnt signaling pathways have been implicated in various physiological functions, such as cell fate determination, cell and tissue polarity, synaptogenesis, dendritic morphogenesis, and axon remodeling. Moreover, abnormal Wnt signaling has been implicated in mood disorder. The relationship between Wnt signaling pathway genes and mood disorders has been reported in several genetic association studies. A study showed that alteration of hippocampal microRNA levels following chronic treatment with mood stabilizers is caused by effectors in the canonical Wnt signaling pathway. Gene expression-profiling of hippocampal subfields has also revealed altered expression of several genes related to Wnt signaling in bipolar disorder patients. Another study supports that the canonical Wnt signaling pathway and related substrates play a role in MDD. Wnt signaling pathway also has been considered relevant to the antidepressant effects, and Wnt2 expression and signaling is a common target of antidepressants and that increased Wnt2 is sufficient to produce antidepressant effects.Moreover, patients with MDD have depressed mood or anhedonia but SSD have not, so differential expression of genes involving in other 51 pathways in MDD may be correlate with the underlying pathological mechanism of the symptom of depressed mood or anhedonia. Our unsupervised hierarchal clustering analysis showed obviously that each disease state exhibited a unique expressed genome signature except the genes involving in MAPK and Wnt pathways, which suggesting that these two diseases may be two different phenotypes in depression spectrum by respective gene signatures. Furthermore, genes differential expression among SSD group, MDD group, and healthy controls allowed us to discriminate among these three groups. It suggested that blood-derived RNA may potentially be used as a diagnostic tool for SSD and/or MDD, as long as the correct subsets of genes are employed. Blood profiling may also allow identification of differentially expressed genes that are involved in the pathophysiology of these disorders. To select the most potential biomarkers for differentiating these three groups, we combined top differential expressed genes from each set of gene expression signatures, then trained and tested the multiple combinatorial gene signatures from pair-wise comparison groups by using support vector machine classifier. Finally 48 gene expression signatures were determined. Samples can be grouped together according to the similarity of the expression levels of these 48 genes which suggested that different levels of gene expression may reflect different disease states. Among differential genes, BDNF, COX5B, GRK6 are the most significantly differential genes.We comprehensively analyzed gene functions and pathway for the candidate biomarkers of SSD and MDD and found that potential biomarkers act in some pathways which have been found associated with function of CNS and implicated in depression, including insulin signaling pathway, signaling by NGF, ErbB signaling pathway and neurotrophin signaling pathway. We also found most of them were not reported the relationship with depression, such as cell surface interactions at the vascular wall, NRAGE signals death through JNK, Rho GTPase cycle, and G alpha signaling pathway, etc. al.Some studies showed that there was a positive association between depressive disorder and insulin resistance due to dysregulation of insulin secretion or insulin receptor signaling. Otherwise, various functions for insulin receptor signaling in the brain have been suggested in normal neurophysiology, such as insulin receptor signaling maybe play a important role in synaptic plasticity and cognitive function,and several lines of work in both laboratory animals and humans suggest that when neurons in cognitive brain regions such as the hippocampus and cerebral cortex do not make enough insulin or cannot respond to insulin properly, everything from very mild memory loss to severe neorodegenerative diseases can result. Dysregulation of insulin secretion or insulin receptor signaling has also been reported in serious mental illnesses, such as Alzheimer's disease. Patients with depression also have some cognitive function problems and maybe have differential expression of genes involving in insulin signaling pathway.It has been suggested that neuronal atrophy or destruction in the hippocampus and cortex is involved in the pathogenesis of depression. The neurotrophin systems modulate neuronal plasticity, inhibit cell death cascades and increase cell survival proteins that are responsible for proliferation and maintenance of central nervous system neurons. Thus the dysregulation of the neurotrophin systems, such as differential expression of genes involving in signaling by NGF (nerve growth factorA) and neurotrophin signaling pathway, may be involved in the pathophysiology of depression.Transgenic mouse experiments have confirmed that the block of erbB signaling pathway will result in the change of OL number and morphology, reducing the thickness of myelin and the transmission rate of CNS axons The results of analysis of the cis-regulaotry elements co-occurring on the promoters of these genes showed that STAT1 and STAT2 factors were detected on five MDD signatures' promoters . Especially, STAT1 mediates the autoimmune and inflammatory functions, and STAT2 mediates the virus protection function. From previous investigation about the immune cell specificity of activation programs induced by a major component of cell-mediated immunity, the transcriptional activators STAT1 were significantly induced in CD4+ and CD8+ T cells, B cells and monocytes The results presented were limited by a modest sample size and required more samples to replicate. Quantitative reverse transcription-polymerase chain reaction were required to exam the expression levels of 48 genes, which were found differentially expressed in our pilot study, in a larger sample of SSD and MDD. Additional studies were required to further explore the roles of these 48 genes in pathophysiology of SSD and MDD.In conclusion, our study demonstrated that SSD and MDD exhibited a unique expressed genome signature with peripheral blood leukocyte, and blood cell–derived RNA may have significant value for performing diagnostic functions and identifying disease biomarkers in SSD and MDD.The study was conducted at the Division of Mood Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine between Jan 2007 and Dec 2009. Outpatients were recruited from the clinic and ward of Shanghai Mental Health Center. All procedures were reviewed and approved by Institutional Review Boards of Shanghai Mental Health Center. Written informed consent was obtained from each subject before any study-related procedures were performed.Inclusion criteria for SSD group were: two or more depressive symptoms for at least 2 weeks with social dysfunction but without depressed mood or anhedonia, and having a total score of 17-item Hamilton Rating Scale for Depression (HRSD-17) from 8 to 16. Patients were included into MDD group who met DSM-IV criteria for MDD and had the total score of HRSD-17 ≥17. Patients were excluded if they had substance dependence, severe medical illness, organic brain disease, pregnancy. Healthy control subjects have a score 7 or lower on the HRSD-17, and did not have any major Axis I disorders , family history of mental disorder or severe physical diseases .For the gene expression microarray analysis, this study enrolled eight drug-free Chinese Han patients with their first episode of subsyndromal symptomatic depression, eight previously untreated patients presenting with their first episode of major depression disorder, and eight healthy controls. All groups were matched with sex and age shown in .All subjects were screened by the Structured Clinical Interview for DSM-IV (SCID) and assessed through HRSD-17 score by two experienced psychiatrists .Total 20 ml venous peripheral blood from fasting patients and healthy controls were collected during 7am to 9am. Peripheral blood lymphocytes were separated by Ficoll gradient centrifugation using Ficoll-PlaqueTM Plus  24 samples were profiled on affymetrix U133 Plus2.0 GeneChip oligonucleotide arrays , which is comprised of more than 22,000 probe sets and can analyze the expression level of 18,400 transcripts and variants . The preparation of cRNA hybridization, signal scanning, data acquisition, and preliminary analysis were performed at the National Engineering Center for Biochip at Shanghai according to the standard protocols recommended by Affymetrix . Raw data generated from affymetrix Human U133Plus2.0 were processed and normalized by RMA method with Gene Spring Software 11.0 , then the values were log2 transformed. Differential gene analysis was preliminarily performed using Welch t test and then P value adjustment under multiple hypothesis testing was implemented with multtest package in Bioconductor under the adjustment method of Bonferroni. We used Welch t test and boost strap resampling approach  to compute t statistics and p values. The threshold for differential expressed genes (DEGs) was chosen as 0.01.http://www.mged.org/Workgroups/MIAME/miame.html. The accession numbers is GSE32280.All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database , as detailed on the MGED Society website To select the smallest size of biomarkers with robust predictive power and fewer potential false positives, more stringent thresholds were used to identify genes with even greater reliability. Firstly, the thresholds for differentially expressed SSD and MDD signatures compared to control and differentially expressed signatures between SSD and MDD were set as 1.0E-4, 5.0E-4 and 1.0E-4 respectively. Alternatively, P values in combination with fold-change values were used to identify potential biomarker genes to limit the likelihood of false positive results. Secondly, these signatures from 3 pair-wise comparisons were ranked according to their adjusted P values and the top N signatures were merged directly . Then, we applied SVM (Support vector machines) on each of candidate expression profiles to search better combination of biomarkers with robust prediction performances . Finally, leave-one-off method was used to validate the biomarkers. Leave-one-off validation involves using a single observation from the original sample as the validation data, and the remaining observations as the training data. This was repeated such that each observation in the sample was used once as the validation data.Standard methods for testing over-representation of a GO category assume that, under the null hypothesis, each gene has equal probability of being detected as DEG  Pathway was analysis using human pathways from KEGG, biocarta, and metabolism pathway databases In order to classify SSD and MDD from healthy control simultaneously, support vector machines (SVMs) was utilized for training and testing on candidate signature expression profiles from signature selection step. SVMs which represents an extension to nonlinear models of the generalized portrait algorithm developed by Vladimir Vapnik is a group of supervised learning methods that can be applied to classification or regression The original SSD, MDD and control problem may be stated in a finite dimensional space, but it often happens that in that space the sets to be discriminated are not linearly separable. For this reason it was proposed that the original finite dimensional space be mapped into a much higher dimensional space presumably making the separation easier in that space. In order to clearly classify SSD and MDD from controls, multiclass SVM were also used in aims to assign labels to instances by using support vector machines. The multiclass approach for conducting this is to reduce the single multiclass problem into multiple binary classification problems. Each of the problems yields a binary classifier, which is assumed to produce an output function that gives relatively large values. In end, polynomial kernel was applied with the best predictive performances for combinatorial gene signatures from the three groups.www.genome.ucsc.edu/). Two thousand bps sequences around TSS for SSD and MDD signatures and biomarkers for classifying three groups  were all considered for in promoter-based de novo motif analysis.Cis-regulatory motifs are essential elements for gene transcription


Human T cell leukemia virus Type 1 (HTLV-1) codes for at least 9 alternatively-spliced transcripts and two major regulatory proteins named Tax and Rex that function at the transcriptional and post-transcriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the Tax/Rex mRNA preceding expression of other viral transcripts. Using transfection of HTLV-1 molecular clones and subcellular RNA fractionation we demonstrated the Rex-dependency of the two-phase kinetics and determined the compartmentalization of the individual mRNAs, showing that over 90% of the HBZ mRNAs were retained in the nucleus. Mathematical modelling revealed the importance of a functional delay of Rex function compared to Tax, which was supported by experimental evidence of delayed accumulation and longer half-life of Rex. These data provided the first evidence for a temporal pattern of HTLV-1 expression and revealed major differences in the intracellular compartmentalization of HTLV-1 transcripts.


Despite the large number of reports attributing the signaling between detached cell cultures to the electromagnetic phenomena, almost no report so far included a rigorous analysis of the possibility of such signaling.In this paper, we examine the physical feasibility of the electromagnetic communication between cells, especially through light, with regard to the ambient noise illumination. We compare theoretically attainable parameters of communication with experimentally obtained data of the photon emission from cells without a specially pronounced ability of bioluminescence.We show that the weak intensity of the emission together with an unfavorable signal-to-noise ratio, which is typical for natural conditions, represent an important obstacle to the signal detection by cells. With the exception of very local physiological events , the signaling through electromagnetic fields—for example light or radio waves—appears to be rare. In a broad sense, this is not true because many organisms communicate by changing the properties of daylight which is reflected from their bodies [Information transfer through signaling molecules is considered the basic principle of communication between cells. Electrical signaling also plays a role, but it is predominantly manifested as a convective electrical phenomena—r bodies ; some orr bodies . Both ofThere are several good reasons to ask these questions. Firstly, well-established photo- and electro-chemical mechanisms make the cellular electromagnetic signaling possible in principle. Why shouldn’t organisms use available physical mechanisms to gain advantage in evolutionary fitness? Secondly, several experimental works attribute the observed effects to some kind of electromagnetic signaling . Few hypA). The results of our consideration indicate that information transfer using this weak light is possible but unfavorable signal-to-noise ratio which occurs under natural conditions represents an important obstacle to signal detection by cells.In this paper, we discuss and analyze the possibilities of cell-to-cell electromagnetic signaling, mainly through light because there are no well established bio-mechanisms known which may serve for emission/perception in other parts of the electromagnetic spectrum. We review modern evidence of cellular signaling through light and summarize preconditions for such signaling. We show that an important limitation for signaling through light is given by the very low intensity of cells’ emissions  which describes signaling between chemically separated cell cultures proposed to be mediated by light or other electromagnetic radiation. Interested readers may find references in several recent reviews-5. AlthoB, C). Observed correlations are then compared to control experiments. The distance between cell cultures in experiments varies most usually between the range of micrometers and millimeters; the maximum reported distance is 4 cm [Present-day experiments which indicate electromagnetic cellular signaling are based on the evaluation of correlation of specified biological parameters between two cell cultures placed in detached neighboring cuvettes or Petri dishes  between two Cacco-2 cell cultures, which were again chemically separated and one of those was exposed to hydrogen peroxide. The correlation in the growth rate between separated populations of Paramecia was shown by Fels [et al.[Albrecht-Buehler disclosed that BHK cells were able to detect the orientation of the other layer of cells, which was separated from the first by a glass film . It was et al.. Two cull.[et al. reported by Fels , who alss [et al..etc. It was proved that the sole closing of the containers with cells indeed reduces the possibility of chemical interaction but it is not the absolute protection. Therefore, the question is, whether the experiments show positive evidence of electromagnetic signaling, rather than lack of evidence for the chemical interaction. If the chemical interaction seems highly unlikely, what makes the electromagnetic interaction likely in an explicit sense? Does the effect of transparency of the barrier provide sufficient evidence?There is no reason to question these results. However, we have to ask whether signaling through the electromagnetic field may provide a defensible explanation of the observed effects. The key uncertainty of the above-mentioned experiments is the degree of chemical separation. Although the authors took measures to minimize the possibility of chemical signaling between the observed cultures, no reported experiment, to our knowledge, shows absolute chemical separation including separated atmospheres, chemically identical surfaces in all controls, First of all, signaling and communication by any medium requires a mechanism modulating the information to be transferred into some physical property of the medium. This is then transmitted to the receiving structure where a mechanism of detection of this modulation has to exist. In order to transfer any information by means of electromagnetic waves, cells must be able to: 1. generate electromagnetic radiation with specific properties, and2. detect the electromagnetic field together with the ability to sense some particular properties of this field.i.e. spontaneous emission of light, is a generally occuring phenomenon in metabolically active systems, even in those without specific enzymatic or protein machinery for bioluminescence [A). Many organic substances also exhibit substantial photo-luminescence, i.e. re-emission of absorbed light. On the other hand, indubitable experimental evidence for non-thermal electromagnetic radiation at lower frequencies is missing [There is no doubt that many types of living cells emit electromagnetic waves with power significantly higher than that corresponding to solely thermal radiation. Non-thermal electromagnetic emission from cells is experimentally proved in the optical range. Auto-luminescence (sometimes termed as Ultra-weak Photon Emission), nescence -16. Whil missing .e.g., those utilizing rhodopsin [Cells may also interact with incident radiation and sense the electromagnetic field. Well proved mechanisms of reception of the electromagnetic field include chains of photo-chemical reactions in the optical range, hodopsin . Besideshodopsin , also othodopsin . Indispuhodopsin  and resohodopsin . Althoughodopsin .As a result, established mechanisms for the generation and perception of electromagnetic waves by cells exist only in the optical range.a. Concerning the cell-to-cell signaling through light, we may only speculate what is the size of the information that is to be transmitted, how is this information coded into the physical properties of the light or what is the coverage of the signal and how is it directed. And many other questions follow. How is the spectrum divided between different streams of information? How many cells/structures can we expect to use, and therefore share, the spectrum? How are interference and crosstalk (i.e. undesired effects between different channels/pathways) suppressed? How is the signaling secured from any possible eavesdropping of predators?As we have shown in previous paragraphs, signaling and bio-communication through light is possible in principle and it might also explain some unforeseen experimental results. So far, however, little is known about the specificity of the generation and reception processes of the light in non-specialized cellsIf we admit the possibility of the idea of signaling between cells through light, we must draw the conclusion that this signaling should be very sophisticated  to cope with the requirements arising from the above-mentioned questions. But even stricter requirements result from the presence of ambient background noise.e.g., luciferases), somewhere between ones and thousands of photons in the visible part of the spectrum are emitted per 1 cm2 per second [15 visible photons per 1 cm2 per second for direct sunlight and decreasing below 103 visible photons per 1 cm2 per second only in very dark environments like caves. This means that the cells which are trying to receive the signal will be exposed more to the useless noise rather then to the signal carrying information. Therefore, the signaling between cells through light shall be possible only in real biological conditions if it can perform despite the presence of ambient noise. Only then it may have some specificity and produce a detectable effect. From the biological point of view, we need to distinguish between two possible regimes of the signaling.Non-specialized cells are hardly able to emit light with an intensity above the level of ambient illumination under natural conditions. Within a colony of standard model cells . Although the distance between the cells in reported biological experiments is only a few centimeters in maximum compared to kilometers in technical single photon experiments, the reduction of the length is offset by increased intricacy of the optical path . It is still uncertain whether single cells are capable of performing any kind of spatial filtering, let alone under such complicated optical circumstances. It is known that only eyes, i.e., organs composed of large number of cells, are able to perform spatial filtering. We can also speculate whether cells can somehow exploit entanglement, angular momentum of photons, or whether they are able to perform modulation of the wave packet of a single photon. However, we still encounter very low probability of detection of modulated signaling photons by the detector cell because the emission level from emitting cells is extremely weak and there is probably no mechanism which can be used for precise spatial alignment between source and detector cells.The first regime does not allow for cooperative behavior between cells within the colony. This regime implies that the intensity of the total emission is just the summation of the individual contributions from all cells, each itself having the possibility of signaling. Therefore, a single cell should be able to emit roughly the same portion of the emission. This portion is supposed to represent the detectable signal. Therefore, a single cell would emit significantly less photons than the entire colony. Any comparison of the measured emissions to the number of cells is rare in literature. Ref.  reports nd. Ref.  reports  systems ,26. Howe21 photons/second/square meter) and very dark circumstances (few photons/second/square meter) in light-tight chambers, deep oceans or caves. Unfortunately, there is no systematic work dealing specifically with the influence of background illumination on the interaction effect. Such investigation might reveal the threshold level of the signaling and help to experimentally estimate noise limitations as well as other parameters of the communication. The majority of reports only state that the experiment was done under moderate indoor illumination or gloom. Since no photo-metric data were provided in those reports, we may only theoretically analyze whether the signaling can take place under similar conditions. Since the wavelength or bandwidth of hypothetical signaling is not known, we will use a general approach of the communication theory and combine it with the established bio-physical data.The second regime of signaling illustrates the eventuality of cooperative signaling. It this case, all cells would cooperate in order to emit detectable signals. The total emission would be either the summation of the coordinated weak contributions or it would be produced by just a few cells from the colony which would communicate with the rest of their neighboring cells by chemical signaling. Regardless of the plausibility of these two options, the total measured intensity of emissions should represent an individual detectable signal. Signaling by means of overall emissions would be far from the single photon regime, yet so far from standard signaling with the intensity of the signal higher than the intensity of the noisy background illumination in the majority of environments. In reality, optical noise may vary between direct Sun-light exposure  is proportional to the channel’s bandwidth, B (in Hertzs), and defined the logarithm of the signal-to-noise ratio as C under conditions of the given signal-to-noise ratio. Strictly speaking, we do not know the size of the information nor the speed required for cellular signaling through light, but we may estimate the plausible range of values of these parameters from the physical feasibility of B and S/N.The limitations of information transmission over a noisy channel are summarized in the famous Shannon’s theorem , a matheB, we may expect that its range should be covered by a single physical mechanism which involves electromagnetic waves. The first reason is that cooperative involvement of different mechanisms—for instance vibrations of polar molecules coordinated with photon emissions—seems unlikelyb. The second reason resides in the fact that the conditions for propagation of electromagnetic waves with extremely different wavelengths may drastically differ. The evolutionary approach would lead cells to occupy those frequencies which naturally have the lowest background noise, i.e. long wavelength radio waves, micro-waves and short ultra-violet wavelengths c. No well described mechanism of reception in cells is, however, known for radio waves. Longer exposure to short UV wavelengths is lethal for living systems. Thermal emission maximum of living systems lies within the infrared range. Therefore, this band should not have any special preference for communication, too, unless cells were able to modulate their thermal emission. So, cells may probably not take the advantage of electromagnetic “silence” on some frequencies. Thus, the maximal bandwidth we may take into consideration is difficult to estimate. However, we may expect that its upper limit will not substantially exceed the frequency extent of the human eye because frequencies higher than visible light have a potentially dangerous impact on living systems. Therefore we use the maximum bandwidth of 800 THz in the following considerations, which is roughly twice the range of the visible spectrum.Concerning the feasible bandwidth, d. The straightforward solution resides in the suppression of the power of the present noise by controlling the environment it lies in. For instance, by the development of special propagation pathways. This might be possible for large organisms, but unrealistic for single cells. Substantial noise suppression might also be achieved by the spatial filtering, i.e. allowing light only from a defined direction to reach the detector. Eyes are capable of performing spatial filtering, but single cells would need extensive machinery to be able to do so as well. Cells would either have to know the positions of other cells or have to selectively scan light from all directions. Both options appear improbable. Nevertheless, signal processing still enables some level of noise suppression if some properties of the specific noise and the signal are known. As we have learned from the signal theory —in general words—it is possible to reconstruct the useful periodical signal hidden in noise by averaging several realizations of the received signal. This can be done because the noise is not correlated between its realizations while the useful signal is. However, this technique has limited efficiency  and it requires a high level of synchronization between the source and the detector. Problems with synchronization and filtering may be overcome by a phase-lock loop. Other possibilities of noise reduction reside in narrow band filtering. These techniques, however, have low efficiency for very low values of the signal-to-noise ratio. Other possibilities of detection, like intensity thresholding or stochastic resonance [S/N by few orders of magnitudes only at very specific intensity levels of noise [e.g.[S/N has not been experimentally reported to our knowledgee. Therefore, we may conclude that the proposed concepts suggesting the possibility of signal-to-noise ratio enhancement by cells are probably not able to provide a general plausible explanation.The number of principles may be, and indeed was, proposed to show how cells could achieve a more favorable signal-to-noise ratioesonance ,29, can’of noise . Some auise [e.g.,32). AnyS/N=10-17) even the very wide frequency spectrum B=1015 Hz is not broad enough to send just the Hello world! message in reasonable time, nor more complicated information at all . For S/N below 10-12, i.e., from a slightly darker towards very dark conditions, the breadth of the required spectrum is comparable to the width of the emission spectra of bio-molecules and the information content per second is sufficient for simple signaling. Therefore, cellular signaling through light under the reported conditions could be possible. Unfortunately, the drastically limited number of emitted photons leads to a very restricted frequency bandwidth. Thus, we can conclude that even though the bio-communication with a highly unfavorable signal-to-noise ratio is possible in principle, it cannot work with such a limited number of photons. This conclusion may be supported by experimental data from photosensitive cells exposed to few-photon coherent signals. Experiments with highly photosensitive rod cells show that detectable responses occur at the minimum of a few tens of photons from a coherent source [Outcomes of these considerations are reflected in Figure t source . If non-In conclusion, we can state that cell-to-cell signaling through light with reported intensities is physically possible only in extremely dark environments where the signal-to-noise ratio is relatively high and the necessary bandwidth is rather narrow. Other possibilities reside in the modulation of the properties of individual photons. However, the existence of mechanisms in biological systems for this purpose is not known. It is also not clear whether the available degrees of freedom of the properties of photons can possibly provide any information transfer without time/spatial filtering and synchronization.So, what do the reports mentioned in the introductory review tell us? Even though the above-discussed limitations support the conclusion that signaling through light may probably not provide credible explanation of the reported effects on the basis of current knowledge, the involvement of light in cell-to-cell signaling cannot be completely disproved. Whether is it light, sound, or volatile compounds, the exact mechanism of interaction between cell cultures mentioned in the introductory section must be identified.In fact, almost all reports mentioned in the introductory review were already driven by the hypothesis that cell signaling is mediated by light, so their authors have not performed measures to undoubtedly exclude other mediators of signaling. Therefore, more sophisticated bio-communication experiments with precise separation of cell cultures are necessary. The design of such an experiment should include isolated atmospheres, identical surfaces of cuvettes and vibration isolation. The barrier with modified spectral transparency which is in fact the only indicator of light involvement in cell signaling, should not come into direct contact with cells in order to prevent any changes in the chemical and/or surface properties of the environment. This means that all cells in the experiment must be in contact with exactly the same surface in order to prevent any physical and chemical effects caused by different surfaces. All relevant parameters like illumination, temperature and chemical properties of the media must be exactly the same for all samples. Needless to say, thorough control experiments are vitally important.In our opinion, an alternative explanation of the observed effects should be sought. While other mechanisms, for instance chemical (volatile compounds) or acoustical (cellular vibrations) pathways, may provide an alternative explanation of the observed effects in certain experimental setups, none of the suggested mechanisms are able to provide a watertight explanation of the observed effects in all the setups. The feasibility of any proposed mechanism would deserve deeper analysis which goes beyond the scope of this paper. Although we advocate that the effects are hardly explicable by light , we hold the view that bio-signaling experiments show us the fascinating ability of cells to detect information. Still, not enough is known about the auto-luminescence properties of living systems. However, a detailed analysis of the properties of photo-emission from cells may bring important data not only for the assessment of hypothetical signaling but also for non-invasive label-free diagnostic purposes.The conclusion we offer is that cellular signaling through light, if it exists as a general phenomenon, employs principles and techniques which are not expected to exist in living cells based on the currently accepted understanding of biophysical processes. So far, it seems that cellular signaling through light (on the level of single cells) is either i.e. the absence of established mechanisms by which electromagnetic fields at levels found in ordinary environments could produce observable changes in biological systems” [1. an example of Foster’s “mechanisms paradox, systems” , or2. is not accomplishable under natural conditions and the reported experiments mentioned in the introductory section should be attributed to another phenomenon.In this paper, we have shown how the idea of cellular signaling through light as a general phenomenon has fundamental limitations given by the ambient noise, which has, in the majority of circumstances, a much higher intensity than the endogenous cellular signal. Cells indeed emit light. However, as a by-product of bio-chemical processes, it has a very weak intensity. This light may bear information about its generation processes, but the detection of such a weak signal would require such mechanisms that would be very difficult, if not outright impossible, to find in biological systems. We may conclude that the signaling between cells which employ electromagnetic waves is possible in principle, but it is very likely limited to very special circumstances or it would require physical mechanisms which are inapplicable in biology or yet unknown.a In this context we use term “specialized cell” to refer to cells which have highly pronounced ability of bioluminescence. The reason for this exclusion is that we are analyzing the cell-to-cell signaling through light as a general phenomenon present in the majority of cells.b Non-linear interactions; however, may broaden the bandwidth.c Under day light conditions, it may be dark only at very short (230 nm - 280 nm) UV wavelengths [elengths . Historielengths , but theelengths . Mainly,d Note that many of proposed principles are tied up with the bandwidth.e Required intensities of the excitation laser light at long wavelengths were, in above mentioned reports [2 which is several orders of magnitudes more than those provided under daylight. We do not know the spectra of photon emission  under exposition by natural daylight because it is not possible to distinguish a photon from a technical source or the Sun from the photon emitted from biological system by a simple measurement. From the auto-fluorescence of tissue [2 to 106 weaker than the intensity of exciting light. reports ,32, abouf tissue  we may eThe authors declare that they have no competing interests.OK conceived the study, performed the analysis and wrote the paper. MC supervised the writing and critically revised the manuscript. Both authors read and approved the final manuscript.


Phosphorylation plays a key role in regulating many signaling pathways. Cell fate decisions in response to extracellular agents, including pathogenic invaders, are commonly mediated by phosphorylation-regulated signaling cascades that transduce signals into stimulus-specific actions, such as changes in gene expression pattern. Here we utilized a novel approach where we infected cells with HTLV wild type and mutant clones and analyzed the phosphorylation status of the cells. For phosphoproteomic analysis the cell lysates were printed onto nitrocellulose membrane slides. Each slide was then probed with one of 360 different antibodies specific against phosphorylated or total forms of signaling proteins. The antibodies were selected to monitor the molecular networks involved in host responses most likely affected by virus exposure, namely survival, apoptosis, inflammation, growth, differentiation, and immune response. This RPMA technique was extensively validated in our previous studies with regard to specificity of antibodies, sensitivity, and accuracy of phosphoprotein detection in cell lysates. Changes in NFkB, SAP/JNK, ERK and AKT and against phosphorylated form of p-38 and PTEN will be discussed.


Objective(s): Few studies have shown the association between HTLV-I infection and coronary artery disease (CAD). HTLV-I has been detected in heart autopsies, particularly in lymphoma\leukemia cases. Mashhad and Neyshabour  are endemic regions for HTLV-I. Therefore, the present study was carried out to evaluate the impact of HTLV-I on CAD in Neyshabourian patients.Materials and Methods: 7590 patients admitted to Razavi and Imam Reza Hospitals (2007-2008) were included in this study. The seroprevalance of HTLV-I infection was determined by the ELISA method and confirmed with the PCR method. Statistical analyses were performed using the SPSS software.Results: Out of the 7590 studied subjects, 564 patients were born and had resided in Neyshabour. The HTLV-I sero-prevalence among these subjects was 13% (n=73). 294 subjects had an abnormal angiography (CAD) and among them 43 (14.6%) were sero-positive for HTLV-I. In the remaining 227 subjects who had a normal angiography, 30 cases were HTLV-I seropositve. The PCR test was performed on 35 cases in order to confirm the presence of infection, which was positive in 31. Regarding the initial population of 294, the rate of PCR-confirmed infection was 10.54%.Conclusion: This sero-prevalence of HTLV-I in subjects with heart complications in Neyshabour was nearly 3 times more than the general population of this city (10.5 % vs 3.4%). However, the results of this study show that in addition to HTLV-I infection, there might be other co-factors leading to the development of heart complications in Neyshabour. Coronary artery diseases (CAD) cause 30% of all mortalities worldwide, making it the most common cause of death in men under the age of 65 and the second most common cause of death among women of the same age.Atherosclerosis is an inflammatory disease with immunologic mechanisms accompanied by metabolic risk factors , 2; it i The HTLV-I retrovirus, which was first diagnosed in 1980 in patients with skin lymphoma can lead to inflammatory disease and is endemic in the Caribbean region and South-West of Japan . Viral aet al study, the prevalence of HTLV-I in Mashhad and Neyshabour  was 2.3% and 3.4%, respectively. According to another study performed in 2003 by Abbaszadegan et al in Mashhad on 28926 blood donors , the prevalence of HTLV-I infection was 0.78% , uveitis, lymphocyte cell alveolitis, Sjogren’s syndrome, thyroiditis, Behcet disease, arthropathy and polymyositis in symptomatic patients. Moreover, this virus is endemic in the Khorasan region and based on the Farid as 0.78% -9.Among the diseases related to HTLV-I, extensive studies have been performed on the pathogenesis of HAM/TSP and various reports have shown that HTLV-I infection in such cases is accompanied by the spontaneous proliferation of T-cells and the production of high levels of pro-inflammatory cytokines such as IL-2, IL-4, IL-6, TNF-α, IL-1α, IL-1β, IFN-γ in serum and CSF and spinal cord injuries in infected patients ,7-11. In the latest review article by Dr. Gessain from the French Pasteur Institute, it has been stated that the main etiology of HTLV-I related diseases might be cardiovascular complications. Therefore, regarding the cardiovascular nature of HTLV-I and the role of inflammation in the pathogenesis of atherosclerosis and coronary artery disease (CAD) and the endemicity of Neyshabour region for HTLV-I infection; in this study the rate of HTLV-I infection and its possible inflammatory effects in patients with coronary vessel involvement was investigated in order to evaluate its risk ratio in causing cardiac disease.Considering the outbreaks of HTLV-I infection in Neyshabour, in a retrospective study, 7590 patients with cardiac symptoms who had undergone angiography in Imam Reza and Razavi hospitals of Mashhad, were enrolled from March 2007 to April 2008. The records of patients from Neyshabour were then studied and those with a positive HTLV-I test before angiography, performed by the ELISA method using an available commercial kit , were determined.For confirming the presence of infection in patients with HTLV-I positive serology tests, after gaining an informed consent, a 5 cc blood sample was obtained from the brachial vein in order to perform the PCR test.Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) using an available commercial kit  and PCR amplification was performed using specific primers for tax (5_- AGGGTTTGGACAGAGTCTT-3_ and 5_-AAGGACCTTGAGGGTCTTA- 3_) and LTR regions (5_-CATAAGCTCAGACCTCCGGG-3_ and 5_- GGATGGCGGCCTCAGGTAGG-3_).Statistical methodsThe collected data were initially entered into the SPSS software, version 16.0. They were then described by tables and statistical indexes. In the end, ANOVA, T-test and Chi-square test were applied in order to analyze the relationship between the studied variables.In this study, 564 patients  with cardiac symptoms from the endemic region of Neyshabour, which has a prevalence rate of 3% for HTLV-I infection in the general population, were assessed. 73 cases (13%) had a positive serologic test for HTLV-I by the ELISA method.P=0.045).The average age of the HTLV-I infected patients was 61.1 years and the average age of the non-infected group was 58.17 years. According to the independent sample test, no significant difference was observed regarding age (P=0.824).In the HTLV-I infected group the average age of the male population was 61 years, whereas it was 61.3 years among females; again showing no statistically meaningful difference regarding sex (P=0.081).In total, 227 cases had a normal angiography, whereas 294 had coronary artery involvement (one to three vessels). The number of subjects with HTLV-I infection among Neyshabourian patients with normal coronary arteries was 30 and in those with coronary artery involvement, it was 43 (14.6%). Based on the Chi-square test no significant difference was revealed in the prevalence of HTLV-I between genders among the cases with coronary artery involvement  and specific env HTLV-I with the PCR method were detected .In this patient, ATL was confirmed as the chronic form with slow progression. This was the first report of isolated lymphoma involvement of the cardiac valve without any other cardiac abnormalities.et al, in a case report in 2002 described a 61-year-old African male from the Caribbean Islands with bowel obstruction and intestinal lymphadenopathy. This patient suffered from non-Hodgkin T-cell lymphoma accompanied by HTLV-I infection. From the clinical study, tachypnea and tachycardia were diagnosed. From echocardiography, a mass in the right atrium originating from the interatrial septum and another mass in the lateral wall of the right atrium protruding into the left ventricle was observed. Cardiac involvement has been repeatedly reported in lymphoma cases. Intra cardiac involvement of non Hodgkin T-cell lymphoma, which responds well to treatment, has only been seen in AIDS patients. This is the first report of cardiac involvement in lymphoma alongside HTLV-I infection  and also unexpectedly another correlation with a history of cardiac disease (relative risk=1.4). Moreover, Masuda reported a 16% prevalence rate for HTLV-I among the 517 patients for which he performed on-pump cardiovascular surgery .The sero-prevalence of HTLV-I infection in subjects with heart complications in Neyshabour was nearly 3 times more than the general population of this city (10.5 % vs. 3.4%). The probable correlation between HTLV-I infection and coronary vessel involvement can be introduced as a risk factor for coronary artery disease with the mechanism of vascular inflammation.


Adult T-cell leukemia (ATL) is a neoplastic disease caused by Human T-cell leukemia virus type 1 (HTLV-1). ATL cells possess a CD4+CD25+ phenotype, similar to that of regulatory T cells (Treg). The HTLV-1 bZIP factor (HBZ), which is consistently expressed in ATL, has a critical role in the development of ATL and HAM/TSP. In the present study, we found that HBZ enhanced TGF-beta/Smad transcriptional activity in a manner dependent on p300. Co-immunoprecipitation assay confirmed that HBZ interacted with Smad3, and formed a ternary complex with Smad3 and p300. In the presence of HBZ, the interaction between Smad3 and p300 was enhanced. The N-terminal LXXLL motif of HBZ was essential for HBZ-mediated TGF-beta signaling activation, while Smad3 interacted with HBZ through its C-terminal MH2 domain. Furthermore, physiological level of HBZ could rescue the repressed TGF-beta responses by Tax. We also found that HBZ activated transcription of the Foxp3 gene through its Smad site. Our study shows that HBZ enhances TGF-beta signaling while Tax suppresses this pathway, and this enhancing activity leads to transcription of Foxp3 gene.


Detection of HTLV infection continues to be important to control spread of human retroviruses and to protect safety of blood supply. The ARCHITECT rHTLV-I/II assay is a chemiluminescence-based immunoassay, which utilizes HTLV-I, and HTLV-II recombinant antigens and peptides in a double-antigen-sandwich assay format on the Abbott ARCHITECT automated platform. Precision, sensitivity and specificity of the ARCHITECT rHTLV-I/II assay is being presented.Assay performance was evaluated on confirmed HTLV-I/II positive specimens and specimens from German Blood Donors and hospitalized patients. External performance evaluation was conducted at two different sites in Portugal. Murex HTLV-I/II EIA was used as comparator assay and INNO-LIA or MP diagnostics HTLV Blot were used for confirmation.The ARCHITECT rHTLV-I/II assay detected all HTLV-I and HTLV-II specimens (N=406) in accordance to the Murex assay. Analytical sensitivity was equivalent to Murex based on end-point-dilutions. Specificity on HTLV-I/II blood donors was 99.95% , SD to cut-off was 15.6. Specificity on a diagnostic population was 99.86% . Precision for samples (1-6 S/CO) was 3.98-4.31%. Presented data support selected cut-off multiplier-value of 0.25 for the ARCHITECT rHTLV-I/II assay. Results from the Receiver-Operating-Curve showed a 99.98% area under the curve which is indicative of high separation power between negative and positive populations.This study demonstrated excellent sensitivity and specificity of the ARCHITECT rHTLV-I/II assay. The assay is suitable for both blood screening and diagnosis of HTLV infections and therefore reduces risk of transfusion transmitted HTLV and improves safety of blood supply.


MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations  that affect MPZ expression.Myelin protein zero (MPZ) is a critical structural component of myelin in the peripheral nervous system. The in vitro and in vivo enhancer assays to study human non-coding MPZ variants.We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish.Our efforts revealed a variant within the first intron of MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.This is the first reported  MPZ protein-coding and splice-site mutations cause demyelinating Charcot-Marie-Tooth disease type 1B (CMT1B) MPZ alleles in patients with CMT1B suggests that there is a threshold in the amount of MPZ required for proper myelination Mpz-knockout allele develop a late-onset peripheral demyelinating neuropathy MPZ transcription.Myelin protein zero (MPZ) is an integral membrane glycoprotein and a major structural component of peripheral nervous system myelin. MPZ has cell-adhesion properties and forms tetramers that are thought to bind other tetramers on opposing membranes, thus holding concentric layers of myelin intact MPZ transcription involves at least two previously described regulatory elements: a proximal promoter and an enhancer located in the first intron MPZ expression are SRY-box 10 (SOX10) and early growth response 2 (EGR2); indeed, mutations in each of these genes have been shown to cause CMT disease in vitro and in vivo, and that mutagenesis of the SOX10-binding sites within this enhancer reduces binding of these transcription factors MPZ transcriptional regulation. In support of this notion, a CMT-associated mutation has been identified in a SOX10 binding site at the gap junction protein beta 1 (GJB1) locus The regulation of MPZ variants within evolutionarily conserved SOX10- and EGR2-binding sites. In this study, we report a rare human variant that alters the activity of an MPZ enhancer, a finding that has implications for understanding the transcriptional regulation of MPZ and for assessing the functional significance of non-coding variants within genes implicated in human inherited neuropathies.We sought to identify and study multi-species conserved sequences (MCSs)] within the MPZ gene. First, we generated and collected sequences of the genomic region encompassing MPZ from multiple species, generated a multi-sequence alignment, and then analyzed that alignment for highly conserved segments. Second, we examined this genomic region on the UCSC Human Genome Browser MPZ-MCS1, MPZ-MCS2, MPZ-MCS3, and MPZ-MCS4  in 192 individuals, including 69 patients diagnosed with demyelinating peripheral neuropathy. These efforts revealed one novel variant (c.126-1086T>A) in a heterozygous state within MPZ-MCS3 , and we did not detect it in a panel of 514 chromosomes from a North America population. Analysis of additional family members revealed that c.126-1086T>A was inherited from the patient's father. However, the father was not available for clinical evaluation, therefore we are unable to conclude whether or not c.126-1086T>A segregates with disease. Examination of our multi-sequence alignment  and thatMPZ (MPZ promoter (MCS2) and intronic enhancer (MCS3) in vitro and in vivoMPZ expression.We tested each of the four conserved non-coding segments at MPZ  for theiMPZ . This inMPZ , transfein vitro enhancer activity of MPZ-MCS3, we cloned the wild-type and variant alleles (both forward and reverse orientations) upstream of a minimal promoter (pE1b) responsible for directing luciferase expression, transfected the resulting reporter constructs into cultured Schwann (S16) cells and oligodendrocytes (OliNeu) cells, and measured the luciferase activity relative to an internal renilla control. These studies revealed that the wild-type MPZ-MCS3 allele enhances luciferase expression by ∼31.5- and ∼7.2-fold (compared to a control reporter-gene construct lacking a cloned insert) in the forward and reverse orientations in S16 cells, respectively . Importantly, these data are consistent with previously-reported analyses on a similar construct with deleted SOX10 binding sites MPZ-MCS3 enhancer activity in OliNeu cells and found similar results as in S16 cells; the c.126-1086T>A variant is associated with a 32–42% reduction of in vitro enhancer activity, slightly less than observed in the S16 cells  cells with, or without, co-transfection of a construct expressing SOX10 , further suggesting that SOX10 specifically acts upon MPZ-MCS3. Interestingly, at each concentration of SOX10 expression construct, the mean value for the enhancer activity of the c.126-1086T>A allele (Var) was lower than that for the wild-type (Wt) allele  allele ; howeverMPZ-MCS3. Briefly, 32P-labeled double-stranded oligonucleotides  containing the SOX10-binding consensus sequence within MPZ-MCS3 were incubated with nuclear extracts from S16 cells ), and then separated on a polyacrylamide gel. The probe containing the wild-type sequence appears to bind a single specific nuclear protein, while the probe containing the v c.126-1086T>A variant binds an additional nuclear protein  in the offspring of multiple independent founder transgenic-zebrafish for each construct.We further studied the function of mpz expression during zebrafish development—mpz is expressed in a dynamic spatial and temporal fashion from early stages in the lateral tegmentum, lens vesicles, optic nerves, hindbrain and anterior spinal cord mpz expression in zebrafish has been reported to be restricted to the CNS during early development Our analyses revealed strong EGFP expression in the telencephalon, midbrain tegmentum, lenses, and hindbrain . At lateMPZ-MCS3 displayed markedly reduced EGFP expression in the forebrain, midbrain, and hindbrain  in a previously described SOX10-binding site MPZ. We characterized this variant in greater detail using a number of methods, including human population screening, comparative sequence analysis, in vitro reporter-gene assays in cultured Schwann and oligodendrocyte cells, electromobility shift assays (EMSAs), and in vivo reporter-gene expression in zebrafish. Our data indicate that the c.126-1086T>A variant resides at a nucleotide position that is highly conserved among mammals, is associated with decreased enhancer activity and altered nuclear-protein binding, and is situated in a genomic segment that directs reporter-gene expression in relevant tissues in developing zebrafish.In this study, we show that a naturally-occurring, rare, non-coding nucleotide variant modulates the activity of an e.g., mouse) harboring c.126-1086T>A MPZ will be necessary to clarify the potential pathogenic role, if any, of this variant. However, our findings indicate that it would be prudent to screen these (as well as other) MPZ non-coding sequences for functional variants in human populations.We were unable to clinically evaluate the transmitting parent (father) of the patient carrying this variant (BAB1733). Therefore, the c.126-1086T>A variant was not implicated in disease onset in humans. Thus, the development of a relevant vertebrate model  directs reporter-gene expression in cultured Schwann and oligodendrocyte cells, does not display enhancer activity in SOX10-negative cells, is responsive to SOX10, and is bound by a nuclear protein—likely SOX10, which has been shown to bind this region in previous studies MPZ represents a rare, functional variant this likely affects MPZ expression in vivo.Our MPZ-MCS3 directs reporter-gene expression in the hindbrain and spinal column in positions consistent with oligodendrocytes. These data are consistent with the established expression pattern of zebrafish mpz at 48 hpf and later MPZ-MCS3 directs reporter-gene expression in distal regions of the descending spinal motor nerves, lateral line nerve, and sympathetic chain nerves. These findings are consistent with the expression of genes in Schwann cells, which myelinate all peripheral neurons. Interestingly, while MPZ is expressed in mammalian Schwann cells, mpz expression has not been detected in zebrafish Schwann cells up to 10 dpf by whole-mount in situ hybridization in situ hybridization may not be sensitive enough to detect mpz expression in zebrafish Schwann cells. Second, mpz may be expressed at a later stage in zebrafish development, due to gene regulation by other elements  acting in a concerted fashion with the intron-1 enhancer. Third, mpz may not be expressed in zebrafish Schwann cells; in this case, the human wild-type MPZ-MCS3 transgene may direct expression of EGFP because sox10 is expressed in Schwann cells in zebrafish by 30–40 hpf The studies we performed in zebrafish revealed that human MPZ shares little sequence conservation between mammals and zebrafish . Second, MPZ-MCS3 may be functional in mammals, but the orthologous region may not be functional in non-mammalian vertebrates ; thus, MPZ-MCS3 might be a mammalian lineage-specific transcriptional regulatory element. Each of these possibilities could be investigated further by analyzing overlapping segments of the first intron of the zebrafish mpz gene for enhancer activity in appropriate cell culture and in vivo model systems.The first intron of ebrafish ; indeed,in vitro and in vivo assays in an attempt to experimentally establish the functional consequences of such variation. While in vitro reporter-gene assays are relatively routine, they do not provide broad biological insight into the role of the non-coding sequences being studied (or of the variants residing therein). Our use of zebrafish to study the wild-type and variant forms of MPZ-MCS3 nicely illustrates the value of an in vivo assay system, particularly since certain mammalian transcriptional regulatory elements are known to function in zebrafish. However, the zebrafish system also has limitations, such as for studying sequences that function in a mammalian- or human-specific fashion and for capturing quantitative data about reporter-gene expression. For the latter limitation, developing a zebrafish system for quantitatively measuring allelic differences in enhancer activity would be a major step toward improving our ability to characterize the functional consequences of non-coding variants and to establish their roles in human disease.The complete sequencing of the human genome All of the studies performed herein were performed under IRB and ACUC approval from the National Institutes of Health (NHGRI), Baylor College of Medicine, and Johns Hopkins University.MPZ gene. First, MPZ-containing bacterial artificial chromosomes derived from the following species were isolated and sequenced as described MPZ in human , mouse , rat , and zebrafish  were obtained from the UCSC Genome Browser (genome.ucsc.edu). All sequences were analyzed using MultiPipMaker  and 2 ng of purified pCMV-RL vector  were diluted in 25 µl OptiMEM I and then combined with the Lipofectamine-OptiMEM I mixture. The ∼50-µl reactions were incubated at room temperature for 20 minutes and then added to a single well of the 96-well culture plate containing cells. After a 3-hour incubation at 37°C, the medium was aspirated, and normal growth medium was added. The SOX10 expression construct was obtained from Origene .Immortalized rat Schwann (S16) cells and mouse motor neurons (MN1) were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1X glutamine, as previously reported Following a 48-hour incubation at 37°C, cells were washed with 1X PBS and lysed at room temperature using 1X Passive Lysis Buffer (Promega). An aliquot (4 µl) of the resulting cell lysate was transferred to a white polystyrene 96-well assay plate . Luciferase and renilla activities were measured using the Dual Luciferase Reporter 1000 Assay System (Promega) and a model Centro LB 960 luminometer . Each experiment was performed at least 16 times. In each case, the ratio of luciferase to renilla activity and the increase in this ratio over that measured for pLGF-E1b with no insert (indicated as ‘fold increase in luciferase activity’) were calculated. The mean  using 10 units of T4 polynucleotide kinase (Promega). The radiolabeled products (probes) were purified using an Illustra™ Microspin™ G-25 column . The ionizing radiation of each probe was measured using a liquid scintillation counter . Nuclear extracts from S16 cells, prepared using a Nuclear Extraction Kit , were incubated in a 20-µl reaction with or without unlabeled double-stranded oligonucleotides (competitor) in the presence of DNA-binding buffer (Promega) for 10 min at room temperature. Samples were then incubated with the above 32P-labeled probes (17.5 fmol/sample) for 20 min at room temperature. The DNA/protein complexes were separated on a 6% TBE DNA Retardation gel (Invitrogen) at room temperature, dried at 80°C for 1 hr, and visualized using a Fujifilm FLA-5000 image analyzer .Complementary pairs of oligonucleotides (sequences available upon request) specific for the genomic DNA encompassing ence see  were annEGFP-expression constructs (see above) were injected into AB background G0 embryos (n≥200) as described EGFP expression were selected and allowed to mature and subsequently crossed, which permitted germline transmission and better evaluation of EGFP expression. Embryos were imaged using a Carl Zeiss Lumar V12 Stereo microscope with AxioVision software (version 4.5).Zebrafish were raised and bred using standard protocols, including maintaining staged embryos at 28°C 7dpf embryos were anesthetized with tricaine (10 µg/ml) in embryo medium Figure S1MPZ-MCS3 wild-type transgenic zebrafish embryos (7dpf) stained for EGPF (A-K) and acetylated tubulin  and MBP (K and L). Panels A-C and J-K display the lateral view at the PLL ganglion region. Panels D-F represent lateral views showing trunk expression, whereas panels G-I show sympathetic chain expression from the ventro-lateral view; anterior is to the left; white arrowheads indicate PLL; white arrows indicate probable oligodendrocytes and blue arrows point at cells expressing EGFP but not AcT in the sympathetic chain nerves.(9.167 MB TIF)Click here for additional data file.


Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI) as a diet-independent complication of the disease. This is a major concern for patients and their parents, and physicians are often asked about possible options to preserve fertility. Unfortunately, there are no recommendations on fertility preservation in this group. The unique pathophysiology of classic galactosemia with a severely reduced follicle pool at an early age requires an adjusted approach. In this article recommendations for physicians based on current knowledge concerning galactosemia and fertility preservation are made. Fertility preservation is only likely to be successful in very young prepubertal patients. In this group, cryopreservation of ovarian tissue is currently the only available technique. However, this technique is not ready for clinical application, it is considered experimental and reduces the ovarian reserve. Fertility preservation at an early age also raises ethical questions that should be taken into account. In addition, spontaneous conception despite POI is well described in classic galactosemia. The uncertainty surrounding fertility preservation and the significant chance of spontaneous pregnancy warrant counseling towards conservative application of these techniques. We propose that fertility preservation should only be offered with appropriate institutional research ethics approval to classic galactosemia girls at a young prepubertal age. Almost every female patient with classic galactosemia, an inborn error of galactose metabolism, has primary ovarian insufficiency (POI) and is confronted with the struggle that reduced chances of having children presents. PatientGALT gene located on chromosome 9p13. GALT is the second of the three enzymes in the Leloir pathway, the main pathway of galactose metabolism. The incidence of classic galactosemia varies between 1:16.000[GALT gene. A galactose-restricted diet quickly resolves the early signs, but cannot prevent the development of later-onset complications, such as cognitive impairment, neurological sequelae, bone health abnormalities, and, in female patients, POI with subsequent infertility.Classic galactosemia ORPHA79239) is caused by deficient activity of galactose-1-phosphate uridyl transferase (GALT), as a result of mutations in the 39 is cauAlthough POI in classic galactosemia represents a major concern for these patients and/or their parents, there aThe aim of this paper is to discuss the different aspects of fertility loss in female classic galactosemia patients from a multidisciplinary perspective, and to propose recommendations for fertility preservation, taking into account pathophysiology, chances of spontaneous pregnancies and ethical issues. These recommendations are based on our experiences with classic galactosemia patients and the currently available literature.Symptoms of POI differ between affected women, varying from subfertility, to early development of irregular menstrual cycles and infertility, to primary amenorrhea and absence of spontaneous puberty. The cauIn general, POI can be caused by either the formation of a smaller primordial follicle pool or more rapid loss of primordial follicles and therHistological findings show that the ovaries contain a strongly reduced number of follicles at older, but variable ages. Histological findings in adolescents (aged 16 or 17 years) reveal a strongly reduced number of follicles, varying from far fewer follicles than expected for age to almosThere is also evidence for defective follicle maturation, which may result from a paucity of follicles able to respond to stimulation or inability of the follicle to respond to gonadotropins in galactosemia. The few follicles present in the ovaries of classic galactosemia patients are mainly of the primordial type, and maturing follicles are rarely seen,23,26,28Animal studies suggest that galactose might already have a direct toxic effect in fetal life. After feeding pregnant and lactating Sprague–Dawley rats a high galactose diet, the offspring had a significant and striking reduction in the number of small follicles,, a reducAn accelerated loss of follicles during life has also been suggested in several studies. Lai et al. found that the number of apoptotic granulosa cells of maturing follicles was higher in Sprague–Dawley rats fed a high galactose diet than in animals receiving normal chow. The samSome animal studies also point to a compromised maturation. A high galactose diet significantly decreased the number of healthy growing and antral follicles, whereas the number of primordial or total atretic follicles was not affected, althougBoth case reports of neonates compared to older girls and the animal studies suggest that galactosemia results in increased follicle apoptosis with an accelerated follicle loss of either primordial follicles or maturing follicles as the cause of POI,35,38. IOver the past years it has become increasingly clear that spontaneous pregnancies in classic galactosemia occur, and occur more frequently than previously thought. In a cohort of 22 patients with classic galactosemia and POI, 9 patients tried to conceive, of which 4 succeeded 44 percent). Althoug percentImportantly, fertility preservation data are mainly derived from cancer patients. The ovaries of a girl affected with non-ovarian cancer are healthy when there is a need for fertility preservation. In contrast, the ovaries of classic galactosemia girls are probably already damaged at an early age. Therefore, the success rate of the procedures is probably lower in galactosemic girls. Perhaps the most important issue to discuss before choosing to apply fertility preservation techniques is the fact that fertility preservation does not guarantee future pregnancy. A thorough evaluation including the abovementioned chance of spontaneous pregnancy and the risks and benefits needs to be taken into account. Three fertility preservation procedures currently offered to girls and women in need of fertility preservation are: a. ovarian tissue cryopreservation, b. mature oocyte cryopreservation and/or c. embryo cryopreservation. The latter two techniques require ovarian stimulation with FSH, whereas cryopreservation of ovarian tissue does not.In ovarian tissue cryopreservation, one ovary is harvested through laparoscopy and the cortex tissue is processed into cortical strips and frozen. When the patient is ready to conceive, the tissue is transplanted at either an orthotopic or a heterotopic location in the body. Currently, twenty healthy infants have been born worldwide as a result of autotransplantation of the cryopreserved tissue. All motIn oocyte cryopreservation the patient’s ovaries are stimulated with exogenous FSH, the growing follicles are punctured transvaginally and the oocytes are extracted from the follicles. The metaphase II oocytes are frozen using vitrification, a technique in which high initial concentrations of cryoprotectant and ultra-rapid cooling are used to create a glass-like state in a cell without forming ice crystals. Once the patient desires to conceive, the oocytes are warmed and consequently fertilized in a petri dish using a partner’s or donor’s semen . The procedure of mature oocyte cryopreservation is no longer considered experimental by multiple international reproduction societies, including the American Society for Reproductive Medicine (ASRM). A recenin vitro using a partner’s or donor’s semen. The fertilized oocytes also develop in vitro to the embryo stage and are then cryopreserved by means of vitrification or slow freezing. Once the patient desires to conceive embryos are warmed and implanted in the uterus. The embryo survival rate is approximately 90% when vitrification is used[A third fertility preservation option is embryo cryopreservation, a well-established and successful technique. This procedure consists of ovarian stimulation, transvaginally harvesting cumulus oocyte complexes and extraction of oocytes. The oocytes are fertilized  is used. Pregnan is used.Since all fertility preservation techniques depend on the ovarian reserve, and conIf fertility preservation is not a reasonable option, or if pregnancy does not occur despite fertility preservation, another option is adoption or the use of donor oocytes to achieve pregnancy. The donor can be anonymous or can be a direct donor, such as a mother or sister. Mothers of galactosemic girls frequently propose to donate oocytes for their daughters. It may be necessary to cryopreserve the mother’s oocytes for the future if the patient is not ready for children at the time of donation. The ethical issues resulting from intra familial donation are discussed below.In the future, other fertility preservation techniques might become available. As an alternative for mature oocyte cryopreservation, cryopreservation of oocytes extracted from immature follicles has been evaluated . Another fertility preservation technique that is currently being evaluated and might be an option in the future is freezing and transplantation of the entire ovary including its vascular system,58. AlsoEthical problems arise in all young patients in need of fertility preservation, regardless of the underlying etiology. Girls are often too young to decide about fertility preservation themselves and therefore their parents play a crucial role in this process. One probably cannot predict with certainty how she herself or, in case of the parents, their daughter will think about a pregnancy in the future. The parent’s decision may not ultimately reflect the girl’s wishes when she becomes an adult. In 2005Cryopreservation of ovarian tissue, the most plausible fertility preservation option for this population, is still considered experimental and pregnancy rates resulting from this procedure are uncertain. RecommeIntrafamilial oocyte donation might lead to ethical issues including psychosocial pressure on the donor and recipient, over-attachment of the donor to the offspring, and role confusion for the persons involved, including the resulting offspring. The proAs a consequence of the severe depletion of the follicle pool that probably occurs prenatally or early in childhood in classic galactosemia patients, fertility preservation in this group is only likely to be successful in very young patients. Oocyte cryopreservation after ovarian stimulation will likely not be successful in most women with classic galactosemia. It might be useful in women with a sufficient ovarian reserve who wish to postpone pregnancy. Ovarian tissue cryopreservation, the only available technique at a young age, has several important disadvantages, including the experimental character of the procedure and the reduced chances of spontaneous pregnancy. Therefore, the technique should probably not yet be offered in a clinical setting, but could be considered on research basis.In general, classic galactosemia patients may have below-average cognitive functioning,63-65 anPhysicians confronted with questions regarding POI and fertility preservation in classic galactosemia, should emphasize that pregnancy has been repeatedly reported despite POI and they should not discourage patients to try to conceive spontaneously. Noteworthy, girls with a spontaneous puberty are likely to have higher chances of spontaneous pregnancy. Furthermore the risk of postponing pregnancy needs to be discussed as well, since in galactosemic women, as in any woman, fertility declines with age, lowering the risk of spontaneous conception. However, due to the unknown course of ovarian function and the unknown pathophysiological mechanism of POI in classic galactosemia, decisions about fertility and fertility preservation remain difficult for patients, parents and physicians.Based on the current knowledge about classic galactosemia and its pathophysiology, we recommend that fertility preservation should not be offered to these patients as common practice . If it is part of institutional research ethics approved research it might be taken into careful consideration. Cryopreservation of ovarian tissue in young prepubertal girls is at this moment the procedure of choice. At a later age, estimated success rates are too low since the ovarian damage is probably already too prominent. Trying to achieve spontaneous pregnancy seems to be the best option for older girls and women.Another option for classic galactosemic girls and women might be oocyte donation, both anonymous donation and intrafamilial donation. We currently do not recommend intrafamilial donation by the mother, as mentioned above. If in a special situation oocyte donation by the patient’s mother is considered, psychological screening and extensive counseling are required, according to previously mentioned guidelines.Finally, we recommend engagement of an independent review board in the process of fertility preservation at minimum two points in the process: when the parental decision to preserve their daughter’s ovarian tissue is considered, but also when the patient wishes to use the preserved tissue, as the parental capacity of the patient should be reviewed.• Physicians should emphasize that spontaneous pregnancies occur in women with classic galactosemia, even after POI diagnosis.• If fertility preservation is desired, cryopreservation at an early prepubertal age as a part of approved research currently seems to be the best option.• The ethics committee of the hospital or another independent body should review the parent’s decision before the fertility preservation procedure.• The ethics committee of the hospital or another independent body should be involved in the decision making surrounding the use of the cryopreserved material.• If a patient desires pregnancy, a one-year window for attempting spontaneous pregnancy is advised to avoid unnecessary use of assisted reproductive techniques.• Anonymous or intrafamilial oocyte donation might be another option for classic galactosemia patients if pregnancy does not occur.The authors declare that they have no competing interests.BE, PhD on galactosemia, performed the study and drafted the manuscript. CG helped to draft the manuscript and gave critical comments. She has her PhD on fertility in galactosemia. RG and GD supervised the gynecological issues. JD gave critical comments, on the assisted reproductive techniques. GW coached the ethical aspect of the review. JG assisted us with critical comments as senior geneticist. AB, ET, GB, reviewed the manuscripts several times as galactosemia experts. CW critically assisted us in this study from her Reproductive Endocrine area of expertise. ER, PhD supervisor and lead of the galactosemia research group in Maastricht, designed, supervised and coordinated the study and the final version of this manuscript. All authors have read and approved the final manuscript.


Although most HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either Adult T-cell Leukemia (ATL) or the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To better understand differential gene expression in HTLV-1-associated disease, we examined the mRNA profiles of CD4+ T-cells isolated from ATL, HAM/TSP, AC and non-infected controls. Using genomic approaches, we identified gene signatures that discriminate between HTLV-1 clinical outcomes. Major activated cellular pathways included antimicrobial defense (KLRB1/SPN/SELPLG), innate immune sensing (TRAF3/AIM2/TLR2/TLR4/IKBKG/STAT3), cell adhesion and chemotaxis (CXCR4/CD2/CD63/CD48/CCL14/SPN/CCL13), thus supporting the idea of global immune disruption during HTLV-1 infection. Among the many genes identified, the suppressor of cytokine signaling 1, SOCS1, was upregulated in HAM/TSP and AC. SOCS1 expression positively correlated with high HTLV-1 mRNA load that is characteristic of HAM/TSP patients. SOCS1 inhibited cellular antiviral signaling during HTLV-1 infection by degrading IRF3, an essential transcription factor in the interferon pathway. This novel mechanism of viral evasion of the innate immune response can be directly related to the efficiency of HTLV-1 replication in patients with HAM/TSP. Despite SOCS1 ability to limit antiviral responses, CD4+ T-cells from HAM/TSP patients revealed a striking upregulation of IFN stimulated gene expression, viral host restriction factors and pro-inflammatory genes. We hypothesize that the magnitude of innate immune responses against HTLV-1 during de novo infection may predict clinical outcome; patients mounting a sustained/low antiviral response to HTLV-1 may remain AC with a low probability to progress to ATL, while those displaying an aberrant/high IFN response may progress to HAM/TSP.