After more than half a century of modern psychopharmacology, with billions of dollars spent on antidepressants annually world-wide, we lack good evidence to guide our everyday decisions in conducting antidepressant treatment of patients with major depression. First we did not know which antidepressant to use as first line treatment. Second we do not know which dosage we should be aiming at with that antidepressant. Because more than half of the patients with major depression starting treatment do not remit after adequate trial with the first agent, they will need a second line treatment. Dose escalation, augmentation and switching are the three often recommended second line strategies but we do not know which is better than the others. Moreover, we do not know when to start considering this second line treatment.The recently published multiple-treatments meta-analysis of 12 new generation antidepressants has provided some partial answers to the first question. Starting with these findings, this proposed trial aims to establish the optimum 1st line and 2nd line antidepressant treatment strategy among adult patients with a non-psychotic unipolar major depressive episode.SUN(^_^)D, the Strategic Use of New generation antidepressants for Depression, is an assessor-blinded, parallel-group, multi-centre randomised controlled trial. Step I is a cluster-randomised trial comparing titration up to the minimum vs maximum of the recommended dose range among patients starting with sertraline. The primary outcome is the change in the Patient Health Questionnaire (PHQ)-9 scores administered by a blinded rater via telephone at week 1 through 3. Step II is an individually randomised trial comparing staying on sertraline, augmentation of sertraline with mirtazapine, and switching to mirtazapine among patients who have not remitted on the first line treatment by week 3. The primary outcome is the change in the PHQ-9 scores at week 4 through 9. Step III represents a continuation phase to Steps I and II and aims to establish longer-term effectiveness and acceptability of the above-examined treatment strategies up to week 25. The trial is supported by the Grant-in-Aid by the Ministry of Health, Labour and Welfare, Japan.SUN(^_^)D promises to be a pragmatic large trial to answer important clinical questions that every clinician treating patients with major depression faces in his/her daily practices concerning its first- and second-line treatments.NCT01109693ClinicalTrials.gov:  In JapaBoth pharmacotherapy and psychotherapy have been found to be equally effective in treating major depression  but the There is no question that we need a specific, detailed and appropriate guidelines in the treatment of major depression. However, all the guidelines up to 2008, including the one by the American Psychiatric Association , the oneHowever, in 2009, the research group from Japan, Italy and UK published the results of a systematic review of 117 RCTs (25928 subjects) of 12 new generation antidepressants in the acute phase treatment of major depression . The MetThe MANGA Study observed many statistically significant and clinical meaningful differences among the 12 new generation antidepressants. In terms of efficacy, mirtazapine, escitalopram, venlafaxine and sertraline were among the top four drugs; in terms of acceptability, escitaloporam, sertraline, bupropion and citalopram were superior to the others. The authors concluded that sertraline might be the best choice when starting treatment for moderate to severe major depression in adults because it has the most favorable balance between benefits, acceptability, and acquisition cost.st line treatment, 2nd line treatments must be initiated. Guidelines recommendations for the 2nd line treatment include dose escalation, switching to a different antidepressant possibly from a different class and augmentation [nd line treatment is much scanter than that on the 1st line.Treatment of major depression is not easy because only some 50% respond, i.e. achieve depression severity less than half that at baseline, or only some 30% achieve remission, i.e. return to an euthymic state, after treatment with an adequate dose of antidepressant given for an adequate duration . When paentation ,15. Unfost line antidepressant [First, with regard to dose escalation strategy, three systematic reviews have been published and all concluded that there is no evidence to suggest that dose escalation increases efficacy in comparison with continuing on the same dosage after failure to respond to the 1pressant -18. Nextpressant ,20 both pressant . When dipressant , thyroidpressant  and augmpressant . Other opressant ,25,26 anpressant .nd line strategies against each other than comparing each strategy with staying on the former treatment. For example, the Sequenced Treatment Alternatives to Relieve Depression (STAR*D), which was funded by the NIMH and cost approximately 3 million dollars, examined five switching strategies and four augmentation strategies among the patients who had not achieved remission to the 1st line SSRI treatment but was unable to compare switching versus augmentation as few patients agreed to this randomization [Even less evidence can be found comparing these different 2mization ,29.http://www.medicine.ox.ac.uk/bandolier/index.html), an independent evidence review journal in UK, concluded its review on the MANGA Study by saying, "What the meta-analysis provides is the raw material for the next step, namely creating and testing a care pathway or pathways for depression that provides good results for the largest number of sufferers in the shortest time and at the lowest cost." . This proposed study precisely aims to create and test this optimum care pathway for depression.Review of the literature has revealed that there are indeed many urgent and critical clinical questions that must be answered before clinicians can confidently and competently administer pharmacotherapy for major depression. Urgent because every practitioner encounters these clinical questions almost on a daily basis. Critical because answers to these clinical questions can materially affect the patients' lives. Bandolier  MANGA study showed mirtazapine may be the most effective new generation antidepressant. Due to its less favorable acceptability profile, it was not recommended as the 1st line treatment but, when the latter fails, it is only logical to consider the more effective antidepressant. (ii) Switching is arguably to be preferred over augmentation because combining two drugs may lead to more known and unknown side effects than staying on the same drug.Switching to mirtazapine is a plausible option as the 2nd line treatment for the following reasons. (i) A number of RCTs have provided some evidence to suggest its effectiveness. One small RCT randomly assigned 26 patients who had not responded to SSRI, bupropion or venlafaxine to augmentation either with mirtazapine 15-30 mg/d or with placebo. The remission rates were 46% versus 13% (p = 0.068) [2-adrenergic autoreceptors and heteroreceptors. Mirtazapine also exhibits antagonism to both 5-HT2A, 5-HT2C and 5-HT3 receptors, which results in a net increase in 5-HT1-mediated neurotransmission which is believed to be the primary mediator of efficacy of most antidepressant drugs. Antagonsim of the 5-HT2A receptors has beneficial effects on sexual dysfunction and insomnia, that of the 5-HT2B receptors on anxiety, and that of 5-HT3 on gastrointestinal symptoms, all of which constitute major side effects of SSRIs. (iii) Mirtazapine does not inhibit any liver enzymes and poses very low risk of interaction with other drugs. Sertraline exerts mild inhibition against CYP2D6 and 3A4 but is generally believed to be a safer drug when administered concomitantly with other drugs than many other SSRIs.Mirtazapine augmentation of SSRI is another option as the 2= 0.068) . (ii) Itnd line treatment is when to consider it. As far as practitioners are concerned, this represents just as urgent a clinical question as that of initial titration strategy but, to the best of the authors' knowledge, no RCT has explicitly examined this issue and the guidelines are ambiguous and self-contradictory. For example, the guideline by the American College of Physicians [nd line treatment alternatives [nd line treatment as early as 3 weeks and aimed to examine if considering the 2nd line treatment at this early stage may or may not be beneficial in comparison with continuing the 1st line treatment for 6 more weeks.Another very important clinical question to be answered with regard to the 2ysicians  recommenrnatives . We therst and 2nd line treatments is the continuation treatment following the acute phase treatment. A systematic review has unambiguously demonstrated that discontinuing antidepressants at the end of acute phase treatment can double the relapse/recurrence rates [st and 2nd line treatment strategies is how easy and acceptable it is for patients to continue into the continuation treatment after acute phase treatment, in addition to their effectiveness and acceptability during the acute phase treatment.The last but not least factor to be considered in constructing the optimum treatment strategy for the 1ce rates , and allce rates . It therThe current randomized trial aims to elucidate "pathways for depression that provides good results for the largest number of sufferers in the shortest time and at the lowest cost" (Bandolier 2009). More specifically, the objectives of this trial are to examine the following treatment options among patients with an untreated, non-psychotic unipolar major depressive episode:st line treatment is started with sertraline, which is better as an initial prescription strategy up to 3 weeks in terms of effectiveness and safety (i.e. side effects and treatment continuation), to titrate to the lowest dosage of the effective range or to its highest dosage?(1) When the 1st line treatment at 3 weeks, which is better as acute phase treatment up to 9 weeks in terms of effectiveness and safety, to continue sertraline, to augment sertraline with mrtazapine or to switch to mirtazapine?(2) When the patients do not remit on the 1st and 2nd line treatments is better as acute phase and continuation treatments up to 25 weeks in terms of effectiveness and safety?(3) Which of the above strategies of 1This is an assessor-blinded, parallel-group, multi-centre randomized controlled trial.Participants will be recruited from among those visiting the clinical trial sites according to the following eligibility criteria.1) The participant fulfills criteria for non-psychotic unipolar major depressive episode (DSM-IV) within one month before starting sertraline2) Age between 25 and 75 on the day when sertraline is startedst line drug3) The major depressive episode is the focus of the treatment and the treating physician has judged sertraline to be its appropriate 14) Tolerability to sertaline has been ascertained after 3-16 days of treatment with sertraline 25 mg/d5) The participant is able to understand and sign written informed consent6) The participant is available on the phone for assessment of symptoms and side effects1) Having taken antidepressants, mood stabilizers , antipsychotics, psychostimulants , electroconvulsive therapy, or depression-specific psychotherapies  within one month before starting sertraline2) History of schizophrenia, schizoaffective disorder or bipolar disorder (DSM-IV) as judged by treating physician3) Current dementia, borderline personality disorder, eating disorder or substance dependence (DSM-IV) as judged by treating physician4) Physical diseases which may contraindicate treatment with sertraline or mirtazaapine5) Allergy to sertraline or mirtazapine6) Terminal physical diseases7) Women who are pregnant or breastfeeding 8) Imminent high risk of suicide as judged by treating physician9) Needing non-voluntary hospitalization10) High probability of changing hospital due to relocation etc within 6 months of trial entry11) Cohabiting family members of research staff members of the trial12) Inability to understand written JapaneseNb1) A comprehensive systematic review and meta-analysis has shown that antidepressants increase suicidality in comparison with placebo for people under age 25 but decreases suicidality for people aged 25 or older .2) Both males and females are accepted.3) There is no eligibility criteria for severity of depression as long as the participant meets the diagnostic criteria for major depression. Both outpatients and inpatients are accepted.4) Patients having taken benzodiazepine anxiolytics, tandospirone, hydroxyzine, hypnotic medications, traditional Kampo medications within one month before starting sertraline are not excluded.5) Patients having received psychotherapies other than depression-specific ones  are not excluded.6) Patients with physical diseases that the treating physician judged would not interfere with treatment with sertraline or mirtazapine are not excluded.7) The participant will continue the trial even if his/her diagnosis is changed after trial entry.A participating trial site must fulfill the following eligibility criteria.1) It must have a department of psychiatry or of psychosomatic medicine.2) The principal trial physician and all the participating trial physicians at the site must have understood the study protocol  and have agreed to collaborate.NbA site-visiting CRC will be dispatched to a trial site which1) Is located within one hour at most approximately from the regional centre2) Has more than 100 first-visit patients with major depression per annum3) Has a separate room that the CRC can use for informed consent and that the central assessor can use for telephone assessment.Such trial sites will open, if possible, "a trial clinic" on a certain day of the week to facilitate patients' participation.Each regional centre will recruit collaborating trial sites  in units of 4-5.If the trial site has its own Institutional Review Board, the principal trial physician will seek approval from his/her own IRB and then fax the document of approval to the national centre office. The national central office will examine the document(s) and return the review results to the trial site principal physician by email.If the trial site does not have its own IRB, the principal trial physician will send a proxy form to the IRB at its regional centre and seek approval there.Before the trial site starts recruiting the participants, all the principal trial physician and the participating trial physicians must attend the start-up meeting held either at the trial site or at the national centre. The co-PI and CRC at the regional centre will visit each trial site in order to make sure that the site has finished the preparation and to rehearse the EDC system and blinded central telephone assessment.The overall procedure of the trial is shown in Figures Clinical questions to be answered at each step can be formulated as follows.Patients: Patients with non-psychotic unipolar major depressive episode who had not received treatment for the index episode before starting sertraline and who tolerate sertraline 25 mg/dExposure1: Strategy to titrate sertraline up to the maximum of the effective range, i.e. 25 mg/d -> 50 mg/d -> 100 mg/dExposure2: Strategy to titrate sertraline upt to the minimum of the effective range, i.e. 25 mg/d -> 50 mg/d -> 50 mg/dOutcomes: The primary outcome is the change inPHQ9 scores at week 1 through week 3The secondary outcomes include:1) Change in BDI2 scores at week 1 through week 32) Proportion of remission (4 or less on PHQ9) at week 33) Proportion of response (50% or greater reduction on PHQ9) at week 34) Proportion of successful continuation of the allocated treatment up to week 35) Change in FIBSER at week 1 through week 36) Change inPHQ9 at week1 through week 97) Change in BDI2 at week 1 through week 98) Proportion of remission (4 or less on PHQ9) at week 99) Proportion of response (50% or greater reduction on PHQ9) at week 910) Proportion of successful continuation of the allocated treatment up to week 911) Change in FIBSER at week 1 through week 912) Suicidality as assessed with C-CASA between week 1 and week 913) Manic/hypomanic/mixed episode between week 1 and week 914) Serious adverse events between week 1 and week 9st line treatment with sertralinePatients: Patients whose major depressive episode did not remit (5 or more on PHQ9) at week 3 to the 1Exposure1: Continue sertraline 50 mg/d or 100 mg/d for 6 more weeksExposure2: Augment sertraline with mirtazapine 15-45 mg/dExposure3: Switch to mirtazapine 15-45 mg/dOutcome: The primary outcome is the change in PHQ9 at week4 through week 9The secondary outcomes include:1) Change in BDI2 at week 4 through week 92) Proportion of remission (4 or less on PHQ9) at week 93) Proportion of response (50% or greater reduction on PHQ9) at week 94) Proportion of successful continuation of the allocated treatment up to week 95) Change in FIBSER at week 4 through week 96) Suicidality as assessed with C-CASA between week 3 and week 97) Manic/hypomanic/mixed episode between week 3 and week 98) Serious adverse events between week 3 and week 9Patients: Patients with non-psychotic unipolar major depressive episode who had not received treatment for the index episode before starting sertraline and who tolerate sertraline 25 mg/dExposure1: Strategy to titrate sertraline up to the maximum of the effective range, i.e. 25 mg/d -> 50 mg/d -> 100 mg/d by week 3, then allocated to continue sertraline between week 3 and week 9, then treated at the discretion of the trial physicianExposure2: Strategy to titrate sertraline up to the minimum of the effective range, i.e. 25 mg/d -> 50 mg/d -> 50 mg/d by week 3, then allocated to continue sertraline between week 3 and week 9, then treated at the discretion of the trial physicianOutcome: The primary outcome is the proportion of patients who continue the allocated treatment up to week 25 and are in remission (4 or less on PHQ9) at week 25The secondary outcomes include:1) Proportion of patients who continue the allocated treatment up to week 25 and are showing response (50% or greater reduction on PHQ9) at week 252) Rate of continuation of allocated treatments up to week 253) Change in PHQ9 at week 1 through week 254) Change in BDI2 at week 1 through week 255) Suicidality as assessed with C-CASA between week 1 and week 256) Manic/hypomanic/mixed episode between week 1 and week 257) Serious adverse events between week 1 and week 25st line treatment with sertralinePatients: Patients whose major depressive episode did not remit (5 or more on PHQ9) at week 3 to the 1Exposure1: Continue sertraline 50 mg/d or 100 mg/d for 6 more weeks, then treated at the discretion of the trial physicianExposure2: Augment sertraline with mirtazapine 15-45 mg/d up to week 9, then treated at the discretion of the trial physicianExposure3: Switch to mirtazapine 15-45 mg/d up to week 9, then treated at the discretion of the trial physicianOutcome: The primary outcome is the proportion of patients who continue the allocated treatment up to week 25 and are in remission (4 or less on PHQ9) at week 25The secondary outcomes include:1) Proportion of patients who continue the allocated treatment up to week 25 and are showing response (50% or greater reduction on PHQ9) at week 252) Rate of continuation of allocated treatments up to week 253) Change in PHQ9 at week 4 through week 254) Change in BDI2 at week 4 through week 255) Suicidality as assessed with C-CASA between week 3 and week 256) Manic/hypomanic/mixed episode between week 3 and week 257) Serious adverse events between week 3 and week 25In order to test the feasibility of the study, a pilot study will be run according to this same protocol between December 2010 and October 2011. The pilot study will be a multi-centre study involving:• Nagoya City University Hospital and its affiliated private practices and departments of psychiatry in a general hospital• Kochi Medical School Hospital and its affiliated private practices, departments of psychiatry in a general hospital and psychiatric hospitals• Private practices in YokohamaThe Nagoya site will test recruitment using site CRCs, the Kochi site will test recruitment using site CRCs and direct recruitment by trial physicians, and the Yokohama site will test recruitment using site CRCs dispatched from a commercial site management organization. Feasibility and efficiency of these different recruitment methods will be examined.st 200 patients up to week 25. The pilot study will be reviewed by DSMB who will advise the Steering Committee on the feasibility and safety of the study and on appropriateness of continuing the study. The final decision about whether to continue the study will be made by the Steering Committee. Before continuing the study, the protocol may be amended if necessary and additional trial sites will be recruited.The pilot study will use data of the 1The trial physician and/or site CRC will seek informed consent from a participant at week 1, i.e. 3-16 days after starting sertraline 25 mg/d. The "3-16 days" time frame was chosen to allow two possible visit days to accommodate the participant's schedule at a site where the site CRC makes his/her visits every week. After obtaining the written informed consent, the trial physician or the site CRC makes a face-to-face interview or the central CRC or the central rater will make a telephone interview to assess1) PHQ9 at week 12) FIBSER at week 1These week 1 assessment results will be entered into the EDC along with the complete data on the "Eligibility Form."Eligible participants will be allocated 1:1 to the sertraline 50 mg/d arm and to the sertraline 100 mg/d arm. We will employ cluster randomization by trial site. This cluster randomization will be made by the EDC system. The allocation will use the minimization method adjusting for the number of probable entries as judged by the principal investigator and co-principal investigators (40 or more participants per year vs less than 40 participants per year).We employ cluster randomization for Step I for the following reasons.st line antidepressant up to the minimum effective range or up to the maximum effective range. It is therefore logical to randomize by physician.1) The comparison for Step I is between physician's choice of a strategy to titrate sertraline used as the 12) In reality, because this is an open trial in which the trial physician gradually titrates the dosage taking into account the side effects, having one patient in the sertraline 100 mg/d arm and another in the sertraline 50 mg/d arm may at the same time create contamination in the doctor's decisions. That is, if we randomized by patient, the doctor might tend to stick to his/her personally preferred titration schedule regardless of the individual patient's assignment and reported side effects.3) Likewise, having different doctors with different titration policies within the same trial site might cause unnecessary confusion among the physicians and co-medical staff at the site.4) Asking the participant to undergo individual randomization twice might increase the barrier to participation.5) A number of previous studies have repeatedly reported negligible to very small intra-cluster correlation coefficients ,38.The trial physician will prescribe according to either of the following schedule, depending on his/her own allocated treatment strategy.1) In the 100 mg/d arm, prescribe 50-75 mg/d (once after dinner or before bedtime) for one week at week 1, then prescribe 100 mg/d (once or divided twice per day) for one week at week 22) In the 50 mg/d arm, prescribe 50 mg/d (once after dinner or before bedtime) for one week at week 1, then prescribe the same regimen for one week at week 2The trial physician or the site CRC will ask the participant to fill in BDI2 upon week 2 and week 3 visits. The CRC or the physician will enter the data into EDC.At week 3, the central rater will administer1) PHQ9 at week 32) FIBSER at week 3by telephone. The central CRC will obtain the patient's name and phone number and will keep the rater blind to the name of the clinic and the treatment the participant is receiving. This telephone assessment will normally be conducted in a separate room after the patient arrives at the clinic and before the consultation with the trial physician, so that imminent suicidality may be handled promptly and appropriately according to the "Suicidality Management Manual." If the patient has dropped from the treatment, the telephone call will be made to the mobile phone which he/she has previously registered upon entry into the trial. If strong suicidal wishes are expressed, the central rater will follow the "Suicidality Management Manual."If the patient scores 5 or more on PHQ9 at week 3, as assessed by the central rater, he/she will be randomized for Step II according to the following procedures.If the patient scores 4 or less on PHQ9 at week 3, he/she will continue on the same regimen, and receive the assessments at week 9 and week 25 as planned.The patients scoring 5 or more on PHQ9 at week 3 will be allocated 1:1:1 to the continue-sertraline arm, the mirtazapine augmentation arm, and the mirtazapine switch arm. This randomization will use the minimization method adjusting for (i) site, (ii) whether 50% or greater reduction on PHQ9 is achieved or not, and (iii) whether "moderate" or greater impairment due to side effects is reported on item 4 of FIBSER.The central CRC will enter the necessary data from PHQ9 and FIBSER into EDC. The EDC program will then output "The patient is making steady recovery. Please continue with the same regimen" if the PHQ9 score is 4 or less, and any one of "Augment with mirtazapine. Please add mirtazapine 15 mg/d," "Switch to mirtazapine. Decrease sertraline to half the current dose and add mirtazapine 15 mg/d," or "Continue with sertraline" if thePHQ9 score is 5 or more according to the above randomization. The central CRC will fax the output to the trial physician and the site CRC, so that the physician need not start up the computer every time.If the EDC server is down and/or the trial site cannot use the EDC system for various reasons, the randomisation can be done by calling up the central CRC or the data centre.The details of the three intervention arms are as follows.1) Continue sertraline as specified by Step I cluster randomization. Between week 4 and week 9, sertraline must be kept within the maximum as specified by Step I cluster randomization. If the dosage has not reached this maximum (e.g. 100 mg/d) at week 3, it can be increased to this maximum  between week 3 and week 9.2) Continue sertraline as specified by Step I cluster randomization and add mirtazapine 15 mg/d at bedtime to augment sertraline. After week 4, mirtazapine can be given in 7.5-45 mg/d at bedtime. Between week 4 and week 9, sertraline must be kept within the maximum as specified by Step I cluster randomization. If the sertraline dosage has not reached this maximum (e.g. 100 mg/d) at week 3, it can be increased to this maximum  between week 3 and week 9. Mirtazapine should usually be started at 15 mg/d but can be halved by the treating psychiatrist taking into account age etc of the patient.3) Decrease sertraline to half the current dose and add mirtazapine 7.5-15 mg/d at bedtime in order to switch to mirtazapine. Sertraline must be halved at week 3 and stopped by week 4 or week 5 , so that the patient will receive mirtazapine 7.5-45 mg/d only between week 7 and week 9.The trial physician or the site CRC will continue to ask the participant to fill in BDI2 at every visit between week 4 and week 9. The CRC or the physician will enter the data into EDC.At week 9, the central rater will administer1) PHQ9 at week 92) FIBSER at week 9by telephone. The central CRC will obtain the patient's name and phone number and will keep the rater blind to the name of the clinic and the treatment the participant is receiving. This telephone assessment will normally be conducted in a separate room after the patient arrives at the clinic and before the consultation with the trial physician, so that imminent suicidality may be handled promptly and appropriately according to the "Suicidality Management Manual." If the patient has dropped from the treatment, the telephone call will be made to the mobile phone which he/she has previously registered upon entry into the trial. If strong suicidal wishes are expressed, the central rater will follow the "Suicidality Management Manual."All the participants who have entered the trial are eligible.All the available treatment guidelines for depression recommends that the acute phase treatment, if successful, be continued at least several months. All the treatments between week 9 and week 25 are at the treating physician's discretion. He/she may continue with the same regimen or completely change the regimen. Electroconvulsive therapy and depression-specific psychotherapies can also be administered.The trial physician or the site CRC will continue to ask the participant to fill in BDI2 at every visit between week 10 and week 25. The physician or the CRC will enter the data into EDC.At week 25, i.e. approximately 6 months after trial entry and 4 months after week 9 assessments, the central rater will administer1) PHQ9 at week 252) FIBSER at week 253) History of prescription up to week 25, especially how long the treatment assigned at week 3 was adhered toby telephone. The central CRC will obtain the patient's name and phone number and will keep the central rater blind to the name of the clinic and the treatment the participant is receiving. This telephone assessment will normally be conducted in a separate room after the patient arrives at the clinic and before the consultation with the trial physician, so that imminent suicidality may be handled promptly and appropriately according to the "Suicidality Management Manual." If the patient has dropped from the treatment, the telephone call will be made to the mobile phone which he/she has previously registered upon entry into the trial. If strong suicidal wishes are expressed, the central rater will follow the "Suicidality Management Manual."The following medications are allowed throughout the trial at the discretion of the trial physician.1) Benzodiazepine anxiolytics and hypnotics2) Tandospirone, hydroxyzine3) Gastrointestinal and digestive drugs (except for sulpiride)4) Medications for concurrent physical diseases5) Non-specific psychotherapies (psychotherapies other than depression-specific CBT and IPT), exercise therapy, music therapy, family psychoeducationThrough Step I and Step II, the following treatments are prohibited in principle. However, the patient's safety should be the utmost concern and takes priority over everything else, all appropriate care should be given depending on the patient's condition.1) Antidepressants other than sertraline or mirtazapine2) Antipsychotics3) Mood stabilizers 4) Depression-specific psychotherapies 5) Electroconvulsive therapyThere is no prohibited treatments for Step III.The following cases will be considered deviation from the trial protocol. The participant, however, will not be considered to have dropped out of the study at this stage and will receive the protocol assessments.1) Prohibited concurrent treatments were administered in Step I or II2) The participant was not randomised within the pre-specified time frame for week 3.3) The participant cannot take any sertraline in Step I.4) The participant switches to manic/hypomanic/mixed in Step I.5) The participant turns out to suffer from bipolar disorder, schizophrenia or dementia in Step II.The treating physician is to judge whether 1) through 5) has occurred. If so judged, the physician should immediately notify the site CRC, who will notify the central office.If 1) through 4) occurs in Step I, the randomization for step II will not be made but assessment will continue.If 1) or 5) occurs in Step II, the patient will be analysed as randomized.If the participant meets any one of the following conditions, the trial physician will stop the protocol treatment at his/her discretion. The participant, however, will not be considered to have dropped out of the study at this stage and will receive the protocol assessments.1) The participant wishes to stop the protocol treatment.2) The trial physician judges that it is difficult to continue the protocol treatment because of emergence of serious adverse events (SAE) as defined below.3) The trial physician judges that the risk outweighs benefit in continuing the protocol treatment even when no SAE is reported.4) The participant becomes pregnant and the trial physician judges that the risk outweighs benefit in continuing the protocol treatment.5) The trial physician judges that it is inappropriate to continue the protocol treatment for any other reason.If the participant meets any one of the following conditions, he/she will never be contacted for assessments.1) The participant withdraws consent to receiving protocol assessments, regardless of whether he/she is continuing the protocol treatment.If the trial site meets any of the following conditions, it will be judged "dropout" and will no longer be able to recruit patients. However, the patients who have already entered the study will be followed-up.1) The principal trial physician withdraws his consent.2) No study entry was made within 6 months.3) The Steering Committee judges that it is inappropriate to continue recruitment at this site.The Patient Health Questionnaire was developed in 1999 as a self-report version of the Primary Care Evaluation of Mental Disorders (PRIME-MD) which aims at criteria-based diagnosis of several mental disorders commonly seen in primary care . The depKroenke and his colleagues have provided the following rules of thumb for interpreting the continuous PHQ9 scores .0-4 no depression5-9 mild depression10-14 moderate depression15-19 moderately severe depression20- severe depressionThe minimal clinically important difference, i.e. the smallest difference in score that is considered to be a clinically important intra-individual change, was established to be 5 .The PHQ9 should require less than one minute to fill in for the patient and less than one minute to administer for the clinician . The JapIn this trial, PHQ9 will be administered 5 times at week1, week 3, week 9 and week 25. The central rater will receive training in administering PHQ9 through simulated interviews and will have demonstrated satisfactory reliability. The blindness of the central rater as to the participant's treatment will be assessed by asking the central rater to guess the allocated treatment at week 3, 9 and 25 assessments.BDI2 is a 21-item self-report instrument to measure the severity of depression in adolescents and adults. Its first version was developed in 1961 and slightly amended in 1979 but in 1996 a major revision was undertaken to make the scale more congruent with the modern diagnostic criteria for major depression. In its 40 years of usage, the BDI has become one of the most widely used instruments for detecting possible depression in normal populations and for assessing severity of depression in diagnosed patients .The time frame for evaluation is set to the past 2 weeks including the day of assessment. Each item in the BDI2 has a series of four statements, which describe symptom severity along an ordinal continuum from absent or mild (a score of 0) to severe (a score of 3). The total score therefore ranges from 0 through 63.Good reliability and validity have been reported for the original  as well The original authors proposed the following rules of thumb for interpreting the BDI2 scores 0-13 Minimal14-19 Mild20-28 Moderate29-63 SevereTwo subsequent studies from the US and from Japan basically confirmed these interpretations ,47.A rough guide for interpreting the changes in BDI2 scores may be 0-9 no or slight change, with 5 indicating a minimally important clinical difference10-19 moderate change>= 20 large change.Most patients are comfortable with this 21-item questionnaire and can complete them within 5-10 minutes.In this trial, BDI2 will be filled in by the patient at each visit.FIBSER was originally used in STAR*D as a global rating scale for side effects. This is an observer-rated scale and the Japanese translation has not gone through backtranslation.Continuation of protocol treatment without stopping intervention or stopping assessment as defined above is called "treatment continuation." In Step III, concurrent treatments prohibited in Steps I and II can be used and this will not constitute "treatment continuation."Assessments at week 3, 9 and 25 may be made within the following time frames after week 1.1) ±4 days for assessments at week 1 through 92) ±14 days for assessments after week 9Assessment for week 3 must be made between -4 days to +14 days of the planned date, and that for week 9 must also be made between -4 days to +14 days of the planned date.The data centre will provide the following data monitoring report to the Steering Committee and the DSMB every six months. The chair of the DSMB will assess the data monitoring report, and if he/she finds an ethical problem in the continuation of the trial from the viewpoints of safety or effectiveness, he/she will convene the DSMB and advise the principal investigator to change or stop the study.The data monitoring report will include:1) Progress of the trial regarding trial entry and follow-up2) Implementation of assessments 3) Incidence of serious adverse events 4) Any other problems.Each site will be surveyed within 6 months after the study commencement. The site audit team nominated by the Steering Committee will survey the sites according to the Site Audit Manual. The audit team will report the results to the Steering Committee, which will review them.An adverse event is defined as any unwanted or unintended sign (including laboratory exams), symptom or disease seen in participants of the trial, regardless of the causal relationship with the study intervention.All the protocol interventions in the current trial are within the approved dosage and administration in Japan and will therefore have to follow the Japanese Pharmaceutical Affairs Act.Adverse events will be assessed according to the "Adverse Events Manual" which follows the Japanese Ministry of Health, Labour and Welfare's "Manual for rating the severity of side effects by pharmaceutical products," with an amendment to allow more detailed assessment of suicidality according to Columbia Classification Algorithm for Suicide Assessment (C-CASA) .Adverse events will be classified into:Grade 1: minor side effectsGrade 2: neither major nor minor side effectsGrade 3: major side effects, i.e. side effects that may lead to death or to enduring severe impairment depending on the patient's conditions and circumstancesAll grade 3, and unforeseeable grade 2 adverse events shall be reported to the relevant section of the Ministry of Health, Labour and Welfare as well as to the national centre office. Foreseeable adverse events are judged according to the package inserts of respective drugs. Any grade 3 adverse events that occurred within 30 days of the completion of the protocol treatment shall be reported to the Ministry and the national centre office. The reporting shall be done using the attached "Reporting form on safety of pharmaceutical products."The principal investigator, upon receiving the report, will consult with the trial physician to discuss the course of actions to be taken with regard to the patient in question and also with regard to the study.When a serious adverse event occurs, the trial physician must take all the necessary and appropriate measures to ensure safety of the participant. He/she must also notify the principal investigator immediately. The principal investigator must notify co-principal investigators at all the regional centres within 24 hours, and report to the head of the clinical research institution  through the co-principal investigators. The principal investigator must also notify all the collaborators. The head of the clinical research institution must report to its own IRB and, if it concerns an unforeseeable serious adverse event, must report to the Ministry of Health, Labour and Welfare."A serious adverse event" is defined here as "an adverse event that may lead to death or to enduring severe impairment depending on the patient's conditions and circumstances" and will include:1. Death1.1. All deaths regardless of causal relationship with the protocol treatment, if it is a death during the protocol treatment1.2. Deaths whose causal relationship with the protocol treatment cannot be denied, if it is a death within 30 days after completion of the protocol treatment.2. Life-threatening event3. Event leading to enduring and severe impairment and dysfunctionWhen treatment is required, the trial physician will provide and/or arrange appropriate treatments including hospital admission.Frequent side effectsNausea (18.9%), somnolence (15.2%), dry mouth (9.3%), headache (7.8%), diarrhea (6.4%), dizziness (5.0%) etc.Serious side effectsSerotonin syndrome (unknown frequency), malignant syndrome (unknown frequency), convulsion (unknown frequency), coma (unknown frequency), liver dysfunction (unknown frequency), SIADH (unknown frequency), Lyell syndrome & toxic epidermal necrolysis (unknown frequency), anaphylactoid symptoms (unknown frequency)Frequent side effectsSomnolence (50.0%), dry mouth (20.6%), fatigue (15.2%), constipation (12.7%), increased AST/ALT (12.4%)Serious side effectsSerotonin syndrome (unknown frequency), agranulocytosis/neutropenia (unknown frequency), convulsion (unknown frequency), liver dysfunction/jaundice (unknown frequency), SIADH (unknown frequency)The study will be discontinued by the Steering Committee  upon advice from the DSMB if any of the following conditions is met.1) The causal relationship between any of the protocol treatments and serious adverse events including death is established by this study or by any other study.2) Provision of study drugs becomes impossible for any reason.The data management will be done by the data centre. The electronic data is anonymized in a linkable record, and the participants' names and ID numbers will be recorded only on non-electronic media (e.g. paper notebook) and kept at each trial site.The central CRC will check the progress of all the entered participants every day by use of the EDC and will contact the site CRC or the trial physician should any doubt arise.The data centre will perform similar checks and will contact the central CRC should any doubt arise.The protocol will be published, with TAF as first author. The main papers stemming from Steps I, II and III, especially the one from Step II, will be submitted to a high impact journal. The collaborating researcher has the right to be the first author of these papers in order of their number of recruitment. TAF will remain the corresponding author for all the papers. All the trial principal physicians and the trial participating physicians who have entered more than 10 patients will appear as co-author of at least one paper. Trial principal physicians, trial participating physicians and other members of the Steering Committee, if they do not appear as co-author, will be listed at the end of the article. Such authors may be counted as co-authors in some journals but not in others. The results shall be reflected in treatment guidelines and systematic reviews.The study period of this trial will be between December 2010 and March 2014, with the patient entry period between December 2010 and September 2013.The study period of the pilot study will be between December 2010 and March 2012, with the patient entry period between December 2010 and October 2011.Assuming an intra-cluster correlation coefficient to be 0.05 ,38, withThe clinical question for Step II is the main hypothesis of this trial. Previous studies using PHQ9 in the acute phase treatment of major depression have shown that, on average, the PHQ9 scores will drop from 15 (SD = 5) at baseline to 10 (SD = 6) at end of treatment, with a mean change of 5 (SD = 5) -51. We eOne point difference in the mean change score on PHQ9 corresponds with an effect size of 0.2. This is a small effect according to Cohen's rough rule of thumb for effect size interpretation . HoweverThe sample size will be revisited after completion of the pilot study.Step III represents continuation treatment for Steps I and II, and will therefore be examined as exploratory studies. We therefore will not calculate sample size necessary to detect a significant difference. However, we will calculate the obtained statistical power post-hoc.The pilot study is a feasibility study and needs no sample size calculation. The target sample size is 200. We will perform no statistical analyses looking at the comparison arms at the end of the pilot study, whose participants will therefore be included in the main study unless there is a major change in the study protocol.For Step I, we will compare the sertraline 50 mg/d arm and the sertraline 100 mg/d arm at an individual level. We will test whether the changes in PHQ9 scores at week 1 through week 3 are statistically significantly different between the two arms. Because Step I employs cluster randomization, we will have to take into consideration intra-class correlation coefficients.For Step II, we will test whether the changes in PHQ9 scores at week 4 through week 9 are statistically significantly different among the sertraline continuation arm, the mirtazapine augmentation arm and the mirtazapine switch arm. The null hypothesis that the changes are not different among the treatment arms will be tested by examining treatment effect parameters in the repeated measures analyses of all the eligible subjects in the ITT analysis. We will use random effects model taking into consideration the Step I randomization and Step II randomization factors. We will examine interaction effect of Step I cluster randomisation. The test will be double-sided. The alpha error is set at 0.05 and statistical power at 0.80. We will impute the missing data and carry out sensitivity analyses as necessary.We will perform secondary analyses to supplement our primary analyses and to obtain more elaborate understanding of our clinical questions. The secondary analyses will use models similar to those of the primary analyses. We will calculate relative risks and their 95% confidence intervals for differences in proportions. We will calculate hazard ratios and their 95% confidence intervals for differences in treatment continuation.Details of the statistical analyses will be laid down in the "Statistical Analysis Protocol", which will be prepared by the statistician in the Steering Committee by the time the analyses will be undertaken.We will not perform interim analyses to examine the study hypotheses for two reasons. First, we are not expecting a huge effect size for the planned comparisons and second, it is theoretically likely that trials stopped early for benefit may overestimate the true effect size.However, we will examine the following aspects in order to ascertain the feasibility of the study without revealing the treatment allocation.1) Number of entered subjects per trial site to calculate the number of trial sites and the time necessary to complete the intended study2) The intra-cluster correlation coefficient will be calculated in order to make sure that it is not very different from the one assumed in this protocol. We will re-calculate the sample size if necessary.The analyses for the pilot study will be given in detail in the "Statistical Analysis Plan."All the researchers participating in this trial will place the participants' safety and human rights above everything else and will adhere by the study protocol and the study manual so long as they do not undermine their safety and human rights.All the researchers participating in this trial will abide by the Declaration of Helsinki and its amendments as well as the Ethics Guideline for Clinical Research .Before entry into the trial, the trial physician must explain the following items using the written materials and make sure that the participant has understood the contents of the trial well. Written informed consent will only then be obtained from the participant.1) About clinical trials2) Objectives of the trial3) Name and position of the principal trial physician and names of the participating trial physicians4) Procedures and duration of the trial and what happens after the trial is over5) Advantages to be expected and disadvantages to be anticipated6) Other available treatment options7) The participant can withdraw consent and stop participating in the trial at any time8) There is no disadvantage if the subject does not participate in the trial or stops participating in the trial.9) Medical records that are related to this trial will be seen by study personnel of this trial10) Privacy will be maintained and protected11) Contact address and method when the participant wants more information about the clinical trial or when he/she feels unwell12) Compensation insurance for any health hazards13) Fundings for this trial14) OthersAll the researchers and outsourcers of this trial must strictly protect personal information of the participants in adherence with the Ethics Guideline for Clinical Research and the Private Information Protection Law.Each trial site, each regional centre and the national centre will collect information in anonymized and linkable format. The data centre will not deal with personal information of the participants. The linking information for the participants is strictly managed at each trial site or at the national centre without being computerized, i.e. in paper format.At week 3, week 9 and week 25, the central rater will administer PHQ9 and FIBSER by telephone while being blind to the participant's allocated treatment. The central CRC will arrange this blinded telephone call by obtaining the participant's name and telephone number from each clinic every time. The central CRC will not keep this privacy information at the national centre office.This study has been approved by the Ethics Committee of Kyoto University Graduate School of Medicine, the Institutional Review Boards (IRBs) of Nagoya City University Hospital and of Kochi Medical School Hospital.Each trial site will seek approval of the same protocol if it has its own IRB. If there is no IRB, the trial site will commission its approval to the IRB at Nagoya City University Hospital or at Kochi Medical School Hospital.All the protocol interventions in the current trial are within the approved dosage and administration in Japan. However, because the trial involves random allocation, we will contract a private health insurance to compensate for health hazards that have arisen due to this trial. The contract will be between Kyoto University and Tokio Marine and Nichido Fire Insurance Company. This insurance will cover only death or grade 1 impairment or grade 2 impairment whose causal relationship with the trial cannot be negated. Grades 3-14 impairments will not be covered by this insurance but will be covered by the National Health Insurance, which therefore can incur some copayment. Because all the protocol interventions are within the approved dosage and administration, any health hazards may be object of the National Rescue Scheme for Side Effects. If there is any negligence on the part of the physician, it may be covered with the doctors' liability insurance.The Steering Committee will hold online meetings every two weeks and offline meetings every two months.Principal investigator:Toshiaki A. Furukawa, MD, PhD Co-principal investigators:Tatsuo Akechi, MD, PhD, Norio Watanabe, MD, PhD Shinji Shimodera, MD, PhD (Kochi University Department of Neuropsychiatry)Mitsuhiko Yamada, MD, PhD, Masatoshi Inagaki, MD, PhD Trial statistician:Naohiro Yonemoto, MPH DSMB will consist of three professionals in clinical trials and in psychiatry, who are not involved in this trial: Dr Teruhiko Higuchi , Professor Yoshio Hirayasu  and Akiko Kada . The purpose of DSMB is to check the data monitoring reports prepared by the data centre and make recommendations, where necessary, to the principal investigator.Figure The data centre will be in charge of collecting and managing information independently from the researchers. The data centre will handle site registration, participant registration and allocation, monitor data entry, manage quality assurance of data, manage quality control of data, prepare monitoring reports, and prepare datasets for statistical analyses.The data centre will make monitoring reports on the progress of recruitment, progress of data collection and adverse events to the Steering Committee and DSMB.The data centre will be entrusted a contract research organization to be chosen by public tender bidding.The national centre office will be located within Department of Health Promotion and Behavioral Medicine, Kyoto University School of Public Health. It will have a central rater, a CRC and a secretary.There will be regional centres at Nagoya City University and Kochi University. There will be site CRCs who will make weekly visits to participating trial sites to assist informed consent procedures and to monitor and enter clinical data.The SUN(^_^)D is an assessor-blinded, parallel-group, mutli-centre randomised controlled trial sequentially comparing active treatment options and combinations currently approved for treatment of depression in Japan.The prominent characteristics of the SUN(^_^) D include the following.First, the treatment arms in this trial are based on true clinical uncertainty, because all of them are treatment options currently approved by the regulatory bodies in Japan. In other words, they represent treatment alternatives from which both clinicians and their patients have difficulties choosing at the present moment.Second, the clinical questions to be examined in this trial pertain to urgent and critical decision points for which the world psychiatric and psychopharmacological research community has hitherto failed to provide guidance or answer to.Third, the trial will take place mostly in front-line psychiatric facilities such as private practices and departments of psychiatry of general hospitals from all over Japan, where many if not most of the patients with major depression receive their initial treatment. Because Japan does not have the primary care system as represented by the general practitioners in UK, these are the primary care mental health services in Japan and the current trial is thus expected to have maximum external validity with respect to initial treatment of major depression in Japan.Fourth, several important measures are built in to ensure good internal validity for this pragmatic trial, such as central randomization, blind assessment of symptoms and side effects via telephone and adherence to true intention-to-treat principle through differentiation of discontinuation of protocol treatments and withdrawal from study. Whether we can achieve the last point will depend on whether we can follow up and assess all the patients even when they stop or deviate from the assigned treatments.As discussed in the Introduction, major depression represents the greatest non-fatal burden of disease for the humankind, with commensurate rise in spending on the antidepressants world-wide. We maintain that this must be accompanied by commensurate increase in evidence base to guide their wise clinical administration. For example, the total annual sales of antidepressants amount to 120 billion yen (1.3 billion US dollars), and we would like to advocate that at least 0.1% of this sum be spent on public-domain pragmatic research of their use for mood and anxiety disorders. Many urgent and critical clinical questions can be answered with this research funds only if the research can be simple and pragmatic enough. We hope that SUN(^_^)D can be a template for such future clinical trials, and that it ultimately can provide good evidence to improve the treatment guidelines for depression in the world.5-HT: 5-hydroxytryptamine; BDI2: Beck Depression Inventory-II; C-CASA: Columbia Classification Algorithm of Suicide Assessment; CRC: Clinical Research Coordinator; CRO: Contract Research Organization; CYP: Cytochrome pigment; DALY: Disability-Adjusted Life Years; DSMB: Data Safety Monitoring Board; EDC: Electronic Data Capturing; FIBSER: Frequency, Intensity, and Burden of Side Effects Rating; MANGA: Meta-Analyses of New Generation Antidepressants; MAO: Monoamine oxidase; NICE: National Institute of Clinical Excellence; NaSSA: Noradrenergic and Specific Serotonergic Antidepressant; NNT: Number needed to treat; PHQ9: Personal Health Questionnaire-9; RCT: Randomized controlled trial; SMO: Site Management Organization; SNRI: Serotonin & Noradrenalin Reuptake Inhibitor; SSRI: Selective Serotonin Reuptake Inhibitor; STAR*D: Sequenced Treatment Alternatives to Relieve Depression; SUN ☺ D: Strategic Use of New generation antidepressants for Depression.TAF has received honoraria for speaking at CME meetings sponsored by Astellas, Dai-Nippon Sumitomo, Eli Lilly, GlaxoSmithKline, Janssen, Kyorin, MSD, Meiji, Otsuka, Pfizer, Shionogi and Yoshitomi. He is on advisory board for Sekisui Chemicals and Takeda Science Foundation. He has received royalties from Igaku-Shoin, Seiwa-Shoten, Nihon Bunka Kagaku-sha and American Psychiatric Publication. TA has received speaking fees and/or research funds from Astellas, Astra-Zeneca, Bristol-Meyers-Squib, Daiichi-Sankyo, Dainippon-Sumitomo, Eisai, GlaxoSmithKline, Janssen, Kyowa-Hakko-Kirin, Lilly, Meiji, Otsuka, Pfizer, Sanofi-Aventis, Shionogi and Yakult. NW has received speaking fees and/or research funds from Dainippon-Sumitomo, GlaxoSmithKline, Lilly, Otsuka, Pfizer, Asahi-Kasei and Shering-Plough. SS has received speaking fees and/or research funds from Astellas, Dainippon-Sumitomo, GlaxoSmithKline, Janssen, Lilly, Otsuka, Pfizer, Shering-Plough, Shionogi and Yoshitomi. MY and NY have no conflicts of interest to declare. NY received royalties from Seiwa-Shoten. MI has received a speaking fee from Lilly. KM has received speaking fees from Astellas, Dainippon-Sumitomo, GlaxoSmithKline, Janssen, Lilly, Meiji, Otsuka, Pfizer and Shering-Plough.TAF conceived the study, TAF, MY, MI and NY prepared the original manuscript. TAF, TA, SS, MY, KM, NW, MI and NY participated in the refinement of the protocol. NY is the trial statistician. All authors critically reviewed and approved the final version of the manuscript.


Several lines of evidence indicate altered trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in schizophrenia. Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites. We hypothesized that alterations in AMPA receptor trafficking through the endoplasmic reticulum (ER) may also be altered in schizophrenia. Accordingly, we developed a technique to isolate and measure content of the ER from postmortem brain tissue. We used Western blot and electron microscopy to show that we isolated an ER enriched fraction. We found no changes in the expression of the AMPA receptor subunits, GluR1–4, in the ER from the dorsolateral prefrontal cortex in schizophrenia. These data suggest that AMPA receptor trafficking through the ER is largely intact in schizophrenia. Schizophrenia, a severe mental illness, may be linked to abnormalities of glutamate receptor neurotransmission and expression The AMPA receptor consists of four subunits, GluR1–4, that are typically assembled as dimers into a tetrameric complex in the endoplasmic reticulum (ER) Previous studies have measured the expression of AMPA receptor protein and mRNA at a regional level and found no changes in schizophrenia Based on these findings, we hypothesized that accelerated proximal forward trafficking and decreased ER retention of AMPA receptor subunits may be associated with the pathophysiology of schizophrenia. To further test this hypothesis, we developed a technique to isolate ER from postmortem human brain tissue, characterized this ER fraction, and measured the expression of AMPA receptor subunits in this fraction from subjects with schizophrenia and a comparison group.We used differential sucrose gradient centrifugation to obtain an ER enriched fraction from postmortem brain tissue . In addiEnriched subcellular fractions were further characterized using electron microscopy . The nucWe measured the expression by Western blot analysis of the AMPA receptor subunits, GluR 1–4, in total homogenate samples. Protein expression in the total homogenate was measured relative to the amount of protein loaded. We found no significant changes in GluR1, GluR2, GluR3, or GluR4 in the total homogenate in the schizophrenia group compared to the comparison group. We found no significant correlations between protein expression and age, pH, or PMI. In addition, we found no influence of sex on the expression of these subunits.We examined the expression of the AMPA receptor subunits, GluR 1–4, in the ER fraction . ProteinIn order to assess the validity of our normalization strategy, we also normalized expression of GluR3 and GluR4 to the intra-lane expression of the ER chaperone protein Jena Muenchen antibody 4 (JM4). The expression of JM4 was not changed in either the total homogenate or the ER fraction. We found no significant changes in GluR3 or GluR4 expression normalized to JM4 in the enriched ER fraction (data not shown). Due to technical limitation we were unable to perform these analyses for GluR1 and GluR2. We found no significant correlations between protein expression  and age, pH, or PMI and no influence of sex on protein levels in our enriched ER samples.We have previously reported increased expression in DLPFC in schizophrenia of SAP97 and GRIP1, two proteins involved in the forward trafficking of AMPA receptors from the ER to the synapse Based on Western blot protein characterization and electron microscopy, we have identified contents of the four fractions obtained using our differential sucrose gradient centrifugation protocol. In our ER fraction we found proteins predicted to be in the ER  but no markers from other compartments . The Western blot and electron microscopy profile of supernatant 3 implies that this fraction contains light membranes and cytosol. The presence in this fraction of Grp75, a stress protein typically found in the mitochondrial matrix, may indicate a loss of integrity of mitochondria either during the postmortem interval or processing of the samples. We found that pellet 2 contained post-synaptic densities and intact mitochondria while pellet 1 contained post-synaptic densities and intact nuclei. There is evidence of contamination in these fractions with calnexin and α 1,2-mannosidase, suggesting some breakdown of ER and mitochondrial membranes. Importantly, our fraction of interest, the ER fraction, is generally free from contamination by other subcellular organelles, including mitochondria, nuclei, and postsynaptic densities.Compared to a previous report of ER isolation using immunoprecipitation with magnetic beads, the ER isolation protocol used in this study yields an ER fraction the has been verified to be free from contaminants by Western blot and electron microscopy Previously, we found an increase in GluR1 in early endosomes To assess the validity of our normalization strategy, we also analyzed the expression of GluR3 and GluR4 relative to the same-lane expression of the ER chaperone protein JM4 Similar to other studies using postmortem tissue, there are technical limitations associated with this work. It is conceivable that small changes below our level of detection may be present in schizophrenia and our negative findings are the result of a Type II error. Further, while there was no correlation with expression of any of the subunits with age, all of the subjects included in this study were from an older population, and these findings may not be generalizable to younger patients. The effect of antipsychotic treatment was not assessed in this study because of the small number of available patients off antipsychotic medications at the time of death. It has been shown previously that prolonged treatment of rodents with antipsychotics can alter the expression of some AMPA receptor subunit mRNA and protein expression In summary, we found no significant changes in the expression of AMPA receptor subunits in the ER in DLPFC in schizophrenia. In our previous studies, we examined distal AMPA receptor trafficking in schizophrenia, while in the current study we examined proximal localization of AMPA receptors. With the exception of our earlier GluR1 finding in early endosomes, our findings suggest that proximal and distal subcellular localization of AMPA receptors is largely unchanged in schizophrenia. However, AMPA receptor interacting proteins responsible for the trafficking of the receptors, SAP97 and GRIP1, are increased in schizophrenia. Recent literature has suggested that the AMPA receptor subunits may be variably trafficked within the dendrites Subjects from the Mount Sinai Medical Center brain bank were recruited prospectively and underwent extensive antemortem diagnostic and clinical assessment . ExclusiIsolation of an ER enriched fraction was performed in parallel for each pair of subjects, one control and one schizophrenia subject, using nitrogen cavitation and differential sucrose gradient centrifugation . For each pair of subjects, samples for Western blot were prepared in duplicate with 5–10 µg of protein for each fraction placed in reducing buffer containing β-mercaptoethanol and heated at 70°C for 10 minutes. Protein loaded was consistent for each fraction within subject pairs, but not necessarily between subject pairs. Samples were then run by SDS-PAGE on Invitrogen  4–12% gradient gels, and transferred to PVDF membrane using Bio-Rad semi-dry transblotter . Membranes were blocked in LiCor  blocking buffer for 1 h at room temperature before overnight incubation at 4°C with commercially available antibodies in 0.1% Tween LiCor blocking buffer with antisera dilutions determined empirically . FollowiImage analysis from ER isolation Western blot studies was performed as previously described


Nnat, Klf14, Blcap, Gnas and Ube3a contribute most to the enrichment of TF families. In the mouse, genes that are maternally expressed in placenta are enriched for AP1 binding sites. In the human, we found that these genes possessed binding sites for both, AP1 and SP1.By analyzing the cellular functions of genetically imprinted genes as annotated in the Gene Ontology for human and mouse, we found that imprinted genes are often involved in developmental, transport and regulatory processes. In the human, paternally expressed genes are enriched in GO terms related to the development of organs and of anatomical structures. In the mouse, maternally expressed genes regulate cation transport as well as G-protein signaling processes. Furthermore, we investigated if imprinted genes are regulated by common transcription factors. We identified 25 TF families that showed an enrichment of binding sites in the set of imprinted genes in human and 40 TF families in mouse. In general, maternally and paternally expressed genes are not regulated by different transcription factors. The genes  Genomic imprinting is an epigenetic phenomenon observed in eutherian mammals. For the large majority of autosomal genes, the two parental copies are both either transcribed or silent. However, in a small group of genes one copy is turned off in a parent-of-origin specific manner thereby resulting in monoallelic expression. These genes are called ‘imprinted’ because the silenced copy of the gene is epigenetically marked or imprinted in either the egg or the sperm Igf2 and Igf2r in mouse. Deletion of the paternally expressed Igf2 gene results in intrauterine growth restriction. On the other hand, deletion of the maternally expressed gene Igf2r, results in overgrowth Imprinted genes play important roles in development and growth both pre- and postnatally by acting in fetal and placental tissues The observation that maternally and paternally expressed genes apparently act as antagonists has inspired several evolutionary theories that aim to explain the origin of genetic imprinting under the process of ‘natural selection’ The evolution of a gene regulatory mechanism that silences preferentially one parental allele of a gene implies that paternally and maternally expressed genes experience different selective pressures during evolution. This assumption is supported by the finding that the two groups reveal different patterns of sequence conservation. Whereas the protein-encoding DNA sequences of paternally expressed genes are well conserved among different mammalian species, maternally expressed genes are much more divergent Gnas locus. In the human, genetic and epigenetic aberrations in this region are associated with Albright hereditary osteodystrophy and pseudohypoparathyroidism type 1A or 1B Peg1 and Peg3 genes have a clear behavioral phenotype During postnatal development, genomic imprinting affects endocrinal networks, energy metabolism, and behavior. Prominent examples for the functions of imprinted genes in endocrinal pathways are the imprinted transcripts of the As the phenomenon of genomic imprinting is an important evolutionary facet of mammals with placentas, it is of great interest to identify which sorts of cellular and developmental processes of developing and/or mature organisms are subject to control by imprinted genes. We aimed in this study at characterizing the cellular roles of imprinted genes in an unbiased, data-driven approach. For this, we used the gene annotations of the Gene Ontology (GO) that consists of three structured and controlled vocabularies for the biological processes, cellular components, and molecular functions associated with particular genes. As it is of particular interest to analyze which of these functions are controlled by the sets of maternally and paternally expressed genes, we have also separately analyzed the enrichment of GO terms in these two groups.C15orf2, the expressed allele is unknown since there is no information on the parental origin of the alleles. Copg2, and Zim2 are paternally expressed in the human, but maternally expressed in the mouse. Grb10 exhibits isoform-specific imprinting effects, i.e. there are paternally expressed and maternally expressed isoforms. The other 60 genes have been experimentally classified into paternally and maternally expressed alleles in two equal halves. 25 genes are imprinted in both species, for the remaining imprinted expression was proven only for one of the two species. As control group for the human (mouse) imprinted genes we used all human (mouse) genes that are annotated in the Gene Ontology.Imprinted genes of human and mouse were downloaded from the Catalogue of Imprinted Genes and Parent-of-origin Effects in Humans and Animals (IGC) For analyzing significantly enriched functional categories, we used the functional annotation tool available in the Database for Annotation, Visualization and Integrated Discovery (DAVID) For the most specific GO terms, we ran the same enrichment analysis procedures by using the biological process GO_FAT database instead of using the general GO knowledgebase. GO_FAT is a subset of the full set of GO terms that was established by the DAVID team so that the broadest terms should not overshadow more specific terms. The smaller the p-value, the more enriched is the corresponding GO term in the group of imprinted genes with respect to all human or mouse genes. The map enrichment plugin in Cytoscape Clustering and grouping of the imprinted genes were performed using the DAVID gene functional classification tool. This tool employs a set of fuzzy clustering techniques to classify input genes into functionally related gene groups (or classes). This is done on the basis of the co-occurrence of annotation terms by generating a gene-to-gene similarity matrix based on shared functional annotation. This switches the functional annotation analysis from a gene-centric analysis to a biological module-centric analysis http://bioinfo.vanderbilt.edu/webgestalt/). Using the TF target analysis tool implemented in WebGestalt, we analyzed whether a set of genes is significantly enriched with TF targets (TFT). TFT's are specific sets of genes that share a common TF-binding site defined in the TRANSFAC database The web-based gene set analysis toolkit WebGestalt In this study we addressed the question whether imprinted genes as a group fulfill specific functions in mammalian organisms. For this, we tested if specific GO terms are overrepresented in the group of imprinted genes in comparison to all genes in the human or mouse genome. Of the 41 selected human imprinted genes, 38 are annotated in the GO database that contains in total 14116 human genes. In contrast, all 48 mouse imprinted genes are among the 14219 annotated mouse genes. One should note, though, that many genes are represented by more than one transcript in the GO database.developmental process are linked to numerous more specific terms on the next hierarchical level. Terms that showed an overrepresentation of imprinted genes in both human and mouse with p-values below 0.05 are listed in supplemental First, we analyzed which terms of the Gene Ontology are enriched in the full set of all imprinted genes when compared to the set of all human genes or all mouse genes. We concentrate in this analysis on GO terms that are shared by at least 2 different imprinted genes. In this way, we assume to emphasize those cellular functions that relate to the controlled mono-allelic expression of the set of genes studied here. The terms of the GO database are organized in a tree-like structure where a few general terms such as system development is the term with the lowest p-value. This term is associated with 15 out of the 38 human imprinted genes. This corresponds to a 2.6 fold enrichment in comparison to the annotation frequency in the group of all genes. Cellular processes is the term which is associated with the largest number of imprinted genes in the human: 32 imprinted genes  are associated with this term, whereas this is only the case for 74.6% of all genes. For comparison, the imprinted genes in mouse showed a narrower range of 1.8 and 2 fold enrichment for these two broad terms, and only for system development the p-value is below 0.05. As shown in multicellular organismal development, developmental process, neuron development, system development, and anatomical structure development appear in both species with close to 2-fold enrichment . Only neuron development is 5-fold enriched.In the human, the term system development and cellular processes are rather general terms, we subsequently analyzed the enrichment of terms in the GO_FAT section of the DAVID database. This manually curated section contains only terms that are related to rather specific functions. As shown in CDKN1C and NDN genes. Interestingly, the terms regulation of RNA metabolic process, regulation of transcription, DNA-dependent, and regulation of transcription are the terms that are associated with the largest numbers of human imprinted genes . Moreover, around 8.5% and 10.5% of the examined mouse imprinted genes are involved in the regulation process of phosphorylation and positive regulation of molecular function, respectively. This group includes the imprinted genes Igf2, Ins2, Kcnq1, Htr2a, Grb10, Ndn, Tp73, Impact, Cdkn1c, Zim2, and Plagl1.As terms such as Regulation of RNA metabolic process and the daughter node Regulation of transcription, DNA-dependent are associated with processes involved in the role of RNA synthesis regulation. Some of the encoded proteins are tumor proteins; others are inhibitors of the cell cycle, thus inhibiting division. It is also worth mentioning that the functional term regulation of gene expression by genetic imprinting (this is abbreviated to ‘genetic imprinting’ in the DAVID database) is over-represented as well and is associated with the genes INS, IGF2, and KCNQ1 . These functional associations of IGF2 and KCNQ1 rely on publications reporting how a differentially methylated region in KCNQ1 controls imprinted expression of other genes in the neighborhood IGF2/H19 region of Beckwith-Wiedemann syndrome patients regulation of gene expression by genetic imprinting therefore does not refer to the “property” of the respective gene to be an imprinted gene itself but indeed whether it exerts regulatory function on other genes via genetic imprinting. Consequently, the insulator protein CTCF and the DNA methyltransferase DNMT3A are associated with this term as well.The two GO terms GRB10) is involved in the Negative regulation of transport. This gene interacts with insulin receptors and insulin-like growth-factor receptors GRB10 inhibits tyrosine kinase activity and results in growth suppression, e.g. by suppressing glucose import Organic cation transport and Ion transport describe the regulation of the directed movement of organic cations into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore. The associated mouse imprinted genes Slc22a2 and Slc22a3 are polyspecific organic cation transporters in the liver, kidney, intestine, and other organs.Some functions related to transport are enriched and associated with both human and mouse imprinted genes. For instance, the Growth factor receptor-bound protein 10 . We identified one gene cluster in the human and two clusters in the mouse. The only discovered cluster in human resembles the second cluster in mouse and encompasses zinc finger protein genes such as al tasks . For moual tasks . As mental tasks .nervous system development, organ morphogenesis, and positive regulation of osteoblast differentiation. For the last GO term, the maternally expressed genes even showed a 59.4-fold enrichment (see DLX5 and GNAS) are associated with this term. Therefore, the enormous enrichment likely reflects that positive regulation of osteoblast is so far associated with very few genes in the full genome.In previous studies ment see  althoughorganic cation transport, transmembrane transport, ion transport and organic cation transport. Therefore, not surprisingly, the five maternally expressed genes Kcnk9, Kcnq1, Slca22a2, Slca22a3 and Slca22a18 form a gene cluster that is associated with the same transport-related GO terms. The second gene cluster is formed by TF genes including the maternally expressed genes Klf4 and Zim1  than the maternally expressed genes. Most of them were already present in the over-represented terms for all imprinted genes . Thus weand PEG3 . Hence, Mammalian genes are usually controlled by combinations of different TFs that bind to distinct binding sites in regulatory regions such as the promoters of genes. We were interested in the questions which TFs regulate imprinted genes and if paternally and maternally expressed genes can be distinguished by their TFs. For addressing these questions we applied a similar enrichment analysis (see Methods) to investigate whether binding sites for distinct TFs are enriched in the promoter regions of imprinted genes. This analysis was based on a database of TF targets named Molecular signature Database (MsigDB) Nnat, Klf14, Blcap, Gnas, and Ube3a are the genes that contribute most to the enrichment of transcription factor binding sites.In total, we identified 25 TF families that showed an enrichment of binding sites in the set of imprinted genes in human  . The assKcnq1 and Kcnk9 show an enrichment of heat shock factor 2 (HSF2) binding sites in human and mouse. Similarly, genes that are maternally expressed in placenta, such as Slc22a18, Tfip2, and Phlda2, cluster together in both species. In the mouse, this cluster is characterized by an enrichment of AP1 binding sites, whereas the prominent feature of the human gene cluster is a combination of AP1 and SP1 sites. Finally, UBE3A) was linked to behavioral functions, in contrast to three paternally expressed genes , as well as the paternally expressed genes PEG1 (MEST) and PEG3 that were related both to growth and behavior. Thus, Tycko and Morrison argued that imprinting effects due to either maternally or paternally expressed genes are related to growth whereas behavioral functions are mostly controlled by paternally expressed genes. However, at the present stage, it is unclear if imprinted genes act indeed in the control of behavior, or if the observed behavioral abnormalities in mutant mice are caused by an impaired development of neurons and brain structures.This study analyzed enriched functional annotations of genetically imprinted genes based on the “biological process” tree of the Gene Ontology. In their seminal review Our study did reveal an association of imprinted genes with developmental processes such as organ development in human and mouse. This indicates that these genes function indeed during embryogenesis, but they are not necessarily growth regulating genes. The terms that are related to development in human as well as in mouse are associated with 25% to 44.7% of all imprinted genes in the respective species. Hence, a large proportion of imprinted genes contribute to developmental processes. Imprinted genes are also associated with GO terms that are related to neuronal development. Interestingly, neuronal development is apparently not a function that is restricted to paternally expressed genes. Furthermore, in comparison to developmental functions only a rather small number of imprinted genes (7 genes) show a functional association to the nervous system Several publications have pointed out that imprinted genes play roles in placenta morphology and function. We do not observe a specific association with GO terms that are specifically related to the placenta. Hence, at the first glance our results do not support specific roles in the placenta. However, one should note that many genes that show an expression bias towards the maternal allele in the placenta but not in the embryo have been excluded from this analysis. This was done since it is still under discussion if such biases might be mostly caused by sample contamination with maternal tissue DLX5, GNAS, TP73, PHLDA2, CDKN1C, PPP1R9A, UBE3A) are associated with organ morphogenesis, and more particularly with nervous system development and oesteoblast differentiation. In the mouse, maternally expressed genes form two functional networks that are clearly separated. One is related to transport processes, and includes carrier proteins and channel proteins. Especially transport processes that are a key feature of placenta function are specifically associated with maternally expressed genes in the mouse. The second network consists of terms related to G protein signaling. This network is clearly dominated by CALCR and SLC22A18. For the paternally expressed genes, a functional network is only found in the human. This network consists mostly of terms associated with development, and a few terms that are related to gene regulation. Interestingly, several imprinted genes that encode transcription factors  seem to be key players in this network. Nevertheless, also among the maternally expressed genes are genes that regulate transcription. Thus, regulatory functions are not an exclusive feature of paternally expressed genes.When paternally and maternally expressed genes are analyzed separately, mouse and human show clearly different associations. In the human, several maternally expressed genes  tended to dominate the functional enrichments in the group of paternally expressed genes. In contrast, the enrichments in the group of all imprinted genes were stable even when we removed the well-known genes IGF2, INS, and GRB10.In this context we will briefly consider possible biases in the results obtained. The annotations stored in the Gene Ontology of course only represent a fraction of all knowledge in the original scientific literature and it is impossible to estimate how much we still don't know. It is quite likely that the GO gives a more complete picture about the cellular functions of genes that have been studied intensely compared to the average gene. It is furthermore possible that some of the known imprinted genes such as When grouping the imprinted genes by enriched GO annotations found for at least two genes, we applied the lowest recommended threshold value of 0.3. In future, when more complete functional associations will be available, it remains to be tested whether a higher, more cautious threshold would be advantageous. We found that when applied to the currently available data, this threshold gave a good compromise between coverage and specificity of the obtained results.UBE3A, KLF14, BLCAP, NAP1L5, NNAT, and GNAS, show an over-proportional enrichment of distinct transcription factor binding sites. Interestingly, these genes possess rather diverse functions. For example, UBE3A seems to act in neuronal development, whereas GNAS acts mostly in endocrinal pathways.In the second part of the study, we were interested in the question if functionally related gene groups such as the prominent groups of transcription factors, and transport related proteins, are co-regulated by similar sets of transcription factor families. This is obviously not the case. Interestingly, also maternally and paternally expressed genes are not regulated by distinct sets of transcription factor families. In general, a few genes, i.e. Although imprinted genes appear to be regulated by similar sets of transcription factors in mouse and human, it is difficult to identify a typical transcription factor that regulates imprinted genes. The most prominent factor appears to be SP1. This rather ubiquitous factor might be responsible for the broad tissue spectrum of imprinted genes Plagl1, Gtl2, H19, Mest, Dlk1, Peg3, Grb10, Igf2, Igf2r, Dcn, Gnas, Gatm, Ndn, Cdkn1c and Slc33a4Dlk1, Cdkn1c, Igf2 and Gnas); SP1 regulates three genes  as well as AACTTT_UNKNOWN . We suggest these three transcription factors as candidates that may be responsible for the coregulation of this imprinting network.Varrault and co-workers have recently identified a network of coregulated imprinted genes involving the genes Cdkn1c, Dlk1, Grb10, Gtl2, H19, Igf2, Mest, Ndn, Peg3, and Plagl1) in mouse long-term repopulating hematopoietic stem cells and in representative differentiated lineages. Intriguingly, they found that most of the genes were severely down regulated in differentiated cells. They noticed that their study is the first one that connected imprinted genes that are known to be associated with embryonic and early postnatal growth to the regulation of somatic stem cells. Consequently, they suggested that the balancing forces of growth-promoting paternally expressed genes and of growth-limiting maternally expressed genes may as well play a role in keeping stem cells in the delicate balance of pluripotency. Along these lines, but in the opposite direction, our above finding that the global transcription factors E12 and SP1 play key roles in the regulation of imprinted genes fits to their well-known role in cell differentiation processes Berg and colleagues Table S1Imprinted Gene list. The last column indicates whether the maternal (M) or paternal (P) allele is expressed. P/M means that the gene exhibits species or isoform-specific patterns of imprinting: human COPG2 and ZIM2 were reported to be paternally expressed, whereas these genes are maternally expressed in the mouse. GRB10 encodes maternally, and paternally expressed isoforms. “?” in the imprinting column indicates genes for which imprinting is not confirmed.(DOC)Click here for additional data file.Table S2Enriched GO terms of biological functions for the full set of imprinted genes in human. The table lists the annotation terms, the number of associated genes per each GO term, the ratio of genes annotated with this term relative to the total number of imprinted genes, the p-value and the fold enrichment.(DOC)Click here for additional data file.Table S3Enriched GO terms of biological functions for the full set of imprinted genes in mouse. The table lists the annotation terms, the number of associated genes per each GO term, the ratio of genes annotated with this term relative to the total number of imprinted genes, the p-value and the fold enrichment.(DOC)Click here for additional data file.Table S4Enriched GO terms of biological functions for the maternally expressed genes in human and mouse. The table lists the annotation terms, the number of associated genes per each GO term, the ratio of genes annotated with this term relative to the total number of maternally expressed genes, the p-value and the fold enrichment.(DOC)Click here for additional data file.Table S5Enriched GO terms of biological functions for the paternally expressed genes in Human. The table lists the annotation terms, the number of associated genes per each GO term, the ratio of genes annotated with this term relative to the total number of paternally expressed genes, the p-value and the fold enrichment.(DOC)Click here for additional data file.Table S6The most specific enriched GO terms of biological functions for the paternally expressed genes in human. The table lists the annotation terms, the number of the associated genes per each GO term, percentage of the involved genes to the study genes, the p-value, gene names and the fold enrichment.(DOC)Click here for additional data file.Table S7The enriched Transcription factor target (TFT) families for the full set of imprinted genes in human according to the MSigDB database at significance level 0.01. M and P are the numbers of associated maternally and paternally expressed genes respectively.(DOC)Click here for additional data file.Table S8The enriched Transcription factor target (TFT) families for the full set of imprinted genes in mouse according to the MSigDB database at significance level 0.01. M and P are the numbers of associated maternally and paternally expressed genes respectively.(DOC)Click here for additional data file.Figure S1Heat map for the enriched transcription factor targets in the full set of imprinted genes in human (a) and mouse (b) at p-value of 0.01. Marked in red and blue in the top line are the maternally and paternally expressed genes, respectively.(TIFF)Click here for additional data file.


However, the abundance of the vitamin in the edible portions of the most commonly consumed plants is not sufficient to meet daily requirements. Genetic engineering has proven successful in increasing the vitamin B6 content in the model plant Arabidopsis. The added benefits associated with the enhanced vitamin B6 content, such as higher biomass and resistance to abiotic stress, suggest that increasing this essential micronutrient could be a valuable option to improve the nutritional quality and stress tolerance of crop plants. This review summarizes current achievements in vitamin B6 biofortification and considers strategies for increasing vitamin B6 levels in crop plants for human health and nutrition.Vitamin B6 has an essential role in cells as a cofactor for several metabolic enzymes. It has also been shown to function as a potent antioxidant molecule. The recent elucidation of the vitamin B6 biosynthesis pathways in plants provides opportunities for characterizing their importance during developmental processes and exposure to stress. Humans and animals must acquire vitamin B6 with their diet, with plants being a major source, because they cannot biosynthesize it  Figure 1), namely pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their phosphorylated derivatives. Pyridoxal-5′-phosphate (PLP) is the vitamer of central importance because it is required as a cofactor for over 140 chemical reactions in the cell . Furthermore, the cofactor form of vitamin B6 (PLP) may have positive effects on various forms of cancer by strengthening the immune system and delaying tumor progression . Plants contain multiple B6 vitamers and a substantial fraction of vitamin B6 is present as pyridoxine-5′-β-D-glucoside . While PN, PL, and PM and their phosphorylated derivatives are fully bioavailable from plant food, PN-glucoside is only 50% bioavailable . On the other hand, PN and its glycosylated form are more stable than PL and PLP  are embryo lethal , the supplementation experiments discussed above and a similar study using PL, PM, and PN . This pathway is also referred to as deoxyxylulose 5-phosphate (DXP)-independent , with some of the transgenic lines displaying delayed seed germination and plant growth  promoter . A significant increase in total vitamin B6 content was observed only in seeds (1.2-fold) and the transgenic lines overexpressing PDX2 alone apparently had a higher total vitamin B6 content than the transgenic PDX1 lines. In this study, gene expression was only assessed at the transcript level. However, the protein level also needs to be analyzed to better understand the mechanism behind these observations. On the other hand, the employment of a seed-specific promoter for the expression of both PDX1.3 and PDX2 resulted in a threefold increase of total vitamin B6 in transgenic seeds . In contrast to the work from Herrero and Daub performed with Cercospora genes in tobacco, the latter study reported that ectopic expression of the endogenous PDX genes in Arabidopsis did not affect plant growth and development. However, the choice of PDX1 gene appears to be particularly important considering a more recent report, which describes an at least fourfold increase in total vitamin B6 content in shoots and seeds, respectively using the 35S CaMV promoter . In this study, it was shown that plants overexpressing PDX1.1 at the transcript level also had an increase of the corresponding protein, while PDX1.3 transcript overexpression did not result in an increase at the protein level, suggesting a tighter regulation of this paralog. Indeed, PDX1.3 is known to be an ubiquitination target for protein degradation by the proteasome . In plants, enzymes that are known to be involved include the oxidase PDX3, which transforms PNP or PMP to PLP . Therefore, it is likely that there is cross-talk between de novo and salvage pathways of biosynthesis, which should be taken into account in any biofortification strategy. Notwithstanding, the vitamin increase reported by Efforts to increase vitamin B6 accumulation in plants have so far been focused on genes from the DXP-independent pathway. In a first attempt, the t growth . Moreoveoteasome . Interesoteasome . Howeveroteasome . Overexpargement . Consiste stress . It is ie stress . Howeverl as PLP . In thisP to PLP ; a kinasP to PLP ; a PL reP to PLP  and as yArabidopsis remains to be demonstrated in a crop plant. This also requires a good understanding of the regulatory mechanisms that control genes involved in vitamin B6 biosynthesis and metabolism, regardless of whether a biofortification strategy employs heterologous or homologous expression. A better knowledge of the natural diversity of vitamin B6 content and availability in crop species could also be instrumental in selecting the PDX orthologs providing the highest increase. Comprehensive studies of provitamin A biosynthesis have revealed intra-species genetic mutations linked to differential accumulation of vitamin A precursors that can be exploited to increase vitamin content in crops  the crop has to be widely used and of economic importance; (2) vitamin B6 accumulation in the consumed part of the crop plant should not be constrained by physiological or developmental limitations. Genetic engineering using t intake . Nutritit intake . The genThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.