Rectus sheath block (RSB) is used for postoperative pain relief in patients undergoing abdominal surgery with midline incision. Preoperative RSB has been shown to be effective, but it has not been compared with postoperative RSB. The aim of the present study is to evaluate postoperative pain, sleep quality and changes in the cytokine levels of patients undergoing gynaecological surgery with RSB performed preoperatively versus postoperatively.n = 32) versus postoperatively (n = 32). All of the patients, irrespective of group allocation, will receive patient-controlled intravenous analgesia (PCIA) with oxycodone.This study is a prospective, randomised, controlled , single-blinded trial. All patients undergoing transabdominal gynaecological surgery will be randomised 1:1 to the treatment intervention with general anaesthesia as an adjunct to preoperative or postoperative RSB. The objective of the trial is to evaluate postoperative pain, sleep quality and changes in the cytokine levels of patients undergoing gynaecological surgery with RSB performed preoperatively  cumulative oxycodone consumption at 24 hours after surgery; (2) postoperative sleep quality, as measured using a BIS-Vista monitor during the first night after surgery; and (3) cytokine levels  during surgery and at 24 and 48 hours postoperatively.Clinical experience has suggested that RSB is a very effective postoperative analgesic technique, and we will answer the following questions with this trial. Do preoperative block and postoperative block have the same duration of analgesic effects? Can postoperative block extend the analgesic time? The results of this study could have actual clinical applications that could help to reduce postoperative pain and shorten hospital stays.NCT02477098 15 June 2015.Current Controlled Trials  Multimodal analgesia techniques can be used to provide adequate analgesia for abdominal surgery with midline incision, with the goal of limiting postoperative pain. Each new technique must meet increasing demands for safety and effectiveness. Pain after abdominal surgery has two components, somatic and visceral, both of which are initiated by nociceptor stimulation .Reliance on high-dose opiates to obtain adequate postoperative analgesia has been strongly associated with postoperative nausea and vomiting, sedation and ileus , 3, all Rectus sheath block (RSB) was first introduced into clinical practice in 1899; as described by Schleich, it is a regional anaesthetic technique that involves blocking of the ventral rami of the 7th to 12th intercostal nerves by injection of local anaesthetic into the space between the rectus muscle and posterior rectus sheath to achieve analgesic effects . With thAn early finding that less oxycodone than morphine is needed for visceral pain relief after major abdominal surgery  has beenIn our recent clinical observation, the combination of RSB and patient-controlled intravenous analgesia (PCIA) with oxycodone has been shown to be a potentially better multimodal analgesic technique for patients undergoing transabdominal gynaecological surgery with open midline incision. Thus, this combination treatment might alleviate both somatic and visceral pain. However, it is unknown how this technique influences postoperative sleep quality and cytokine levels.During the first postoperative nights, sleep patterns can be severely disturbed, with marked decreases in total sleep time, slow-wave sleep duration, and rapid eye movement sleep, which are features associated with frequent sleep arousals –29. The RSB is usually performed before surgery because it inhibits reflection of the skin incision and reduces the amount of intraoperative anaesthetic used –38. WhilWe hypothesised that performing RSB after surgery might result in a longer duration of analgesic effects and have a subtle influence on sleep quality after surgery but that it will not decrease the perioperative cytokine levels of patients undergoing gynaecological surgery. The present report will follow the guidelines of the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT).This study is a prospective, randomised, controlled , single-blinded trial. All patients undergoing transabdominal gynaecological surgery will be randomised 1:1 to the treatment intervention with RSB preoperatively or postoperatively. The objective of the trial is to evaluate the postoperative pain, sleep quality and changes in cytokine levels of patients undergoing gynaecological surgery with RSB performed preoperatively versus postoperatively.The primary objective is to compare the interval between leaving the post-anaesthesia care unit (PACU) and receiving the first PCIA bolus injection on the first postoperative night between patients who receive preoperative RSB and those who receive postoperative RSB. The secondary objectives will be to compare the following: (1) cumulative oxycodone consumption at 24 hours after surgery between patients who receive preoperative versus postoperative RSB; (2) postoperative sleep quality, which will be measured using a bispectral index (BIS)-Vista monitor during the first night after surgery , 41; andPatients scheduled for elective transabdominal gynaecological surgery at the First Hospital of China Medical University will be recruited for the study beginning in September 2015. The purpose, procedures, and potential risks and benefits of the study will be explained to the participants, and written informed consent will be obtained.The inclusion criteria are as follows: (1) an age of 18 to 65 years old; (2) scheduled to undergo elective midline incision transabdominal gynaecological surgery for a benign mass; and (3) American Society of Anaesthesiologists (ASA) risk classification I–II.The exclusion criteria are as follows: 1) patient refusal; (2) known hypersensitivity to the study medication (ropivacaine); (3) long-term use of opioids; (4) liver or renal insufficiency; (5) a history of psychiatric or neurological disease; (6) deafness; (7) previous open surgery; (8) regular use of acetaminophen, nonsteroidal anti-inflammatory drugs, corticosteroids, or antiemetics; and (9) a preoperative Pittsburgh Sleep Quality Index (PSQI) global score of higher than 6  and was registered with the Clinical Trials Registry (NCT02477098). All of the participants will provide written informed consent in accordance with the Declaration of Helsinki.All patients requiring transabdominal gynaecological surgery will be randomly assigned via computer-generated sequences placed into sealed envelopes to the following two groups: a general anaesthesia group with preoperative RSB (Group PRE); and a general anaesthesia group with postoperative RSB (Group POST). Treatment allocation will be revealed by opening the envelope on the morning of surgery. All of the patients, in addition to the staff involved in postoperative data collection and analyses, will be blinded to the group allocations. The trial will be monitored by an independent data and safety monitoring organization. The group allocations will not be revealed until the final statistical analysis is completed.All patients will be assessed with the PSQI 1 day before the operation . The PSQ2 at 35–45 mmHg. For maintenance of anaesthesia, sevoflurane  will be used at an end-tidal concentration of 2–2.5 %, and an air-oxygen (FiO2: 50 %) mixture will be adjusted for the maintenance of anaesthesia with intraoperative titration of sufentanil given IV, according to a BIS value of 40 to 60, after intubation in both groups. A 0.05 mg/kg dose of additional cisatracurium will be applied if needed. At the end of surgery, the trachea will be extubated after the return of spontaneous respiration and neuromuscular function, and the patient will then be transferred to the PACU. The patients will receive pain relief  using 5–10 μg titrated sufentanil. Three millilitres of venous blood will be collected for plasma inflammatory cytokine measurements at the PACU.General anaesthesia will be induced as follows: 2 mg/kg propofol given intravenously (IV); 2 mg midazolam given IV; 0.4 μg/kg sufentanil given IV; and 0.2 mg/kg cisatracurium given IV. The patient’s lungs will be ventilated with intermittent positive pressure. The tidal volume will be adjusted to 6–8 ml/kg, and the ventilator rate will be adjusted to maintain end-tidal COBefore surgery (Group PRE) or at the end of surgery (Group POST), a bilateral single-shot RSB will be performed under ultrasound guidance by one anaesthesiologist. The rectus muscle will be imaged with an ultrasound probe in the transverse orientation at the level of the umbilicus. A broadband (5–12 MHz) linear array ultrasound probe  will be used with an imaging depth of 4–6 cm. A PAJUNK  insulated needle  will be introduced a few millimetres from the probe using an in-plane technique at an angle of approximately 45° to the skin. The ultrasound imaging will allow for identification of the rectus muscle and two hyperechoic railway-like lines deep in this muscle (the posterior rectus sheath and peritoneum). Under direct vision, the needle tip will be advanced to the desired position, where 20 ml of 0.5 % ropivacaine hydrochloride will be injected, causing hydrodissection of the rectus muscle away from the posterior rectus sheath. The technique will be repeated on the opposite side. It will be performed by one investigator using a completely aseptic technique.All of the patients, irrespective of the group allocation, will receive PCIA with oxycodone. A PCIA pump  will be set up with a bolus injection of 2 mg every 5 minutes for a maximum of 10 mg every 4 hours without basal infusion. The PCIA pump will be stopped on the third postoperative day, and postoperative nausea and vomiting will be treated with 5 mg tropisetron given IV (on the ward).The patients in this trial will be visited once daily at 24 and 48 hours postoperatively by one of the research personnel. Assessment of sleep quality during the first postoperative night will be performed using the BIS data, and venous blood will be collected.Patients will be excluded from the study for the following reasons: (1) refusal to participate; (2) an exclusion criterion identified during the course of the intervention; and/or (3) the occurrence of a severe adverse event.During the study, patients found to have the following criteria will be removed from the study: (1) the loss of over 500 ml of blood during surgery; (2) an operation time of longer than 3 hours; (3) a violation of the trial protocol; (4) an unacceptable risk of a serious adverse event; or (5) desire to withdraw from the study.The interventional treatment will be administered to patients under standard monitoring in a fully equipped operation room. It is possible that immediate detection of adverse events will occur, in which case they will be treated immediately. However, administration of RSB will be immediately stopped in cases in which the study participant shows significant deterioration. Moreover, the inclusion of each individual patient in the study will be indicated in the electronic hospital information system and hence will be visible to all the physicians and nurses involved in the care of the patient. This visibility will facilitate the reporting of adverse events to the principal investigator. The principal investigator will report suspected unexpected serious adverse reactions to the health authorities.Trained staff will record all of the data and complete a trial-specific electronic case report form (eCRF). At the time of patient inclusion, patient data on demographic characteristics and history of past illnesses will be collected, including age, height and weight, and diagnosis. Intraoperative data on the patients, including the operation start time, durations of anaesthesia and surgery, blood pressure, heart rate, volume loading, amount of sufentanil consumed, and plasma concentrations of IL-1, IL-6, TNF-α, and IFN-γ, will be collected at the following time points: at baseline (before induction), upon arrival at the PACU, and at 24 and 48 hours postoperatively.The time of the first PCIA bolus injection and the amount of cumulative PCIA consumption over 24 hours postoperatively will be recorded.Furthermore, sleep-related baseline data, including the PSQI score for the previous month, will be recorded. Postoperative sleep quality will be measured using the BIS data for the first night after surgery Fig. .Fig. 2Thn = 15.7/ES2 + 1, where ES is the effect size, defined as the difference between the groups divided by the mean of the SD between the groups, with α = 0.05 and power = 0.8. The study is adequately powered, with n = 32 for each group.Sample size was calculated on the basis of the average (mean ± standard deviation (SD)) of the interval between leaving the PACU and their receipt of the first PCIA bolus injection on the first postoperative night calculated in the pilot study . The formula for determining sample size  was n = t test or the Mann-Whitney U test, respectively. Categorical data will be described as a frequency or percentage and will be analysed by the chi-square test. Repeated analysis of variance (ANOVA) will be used to determine cytokine levels within and between the groups. Before statistical testing, each continuous variable will be analysed to determine whether it has a normal distribution using the Kolmogorov-Smirnov test. Nonparametric data will be assessed using Kruskal-Wallis ANOVA or the Mann-Whitney U test as appropriate.Statistical analysis will be performed using SPSS software, version 18 for Windows . A fully specified statistical analysis protocol will be written in an independent manner. Continuous data will be described as the mean (SD or median (25 and 75 % percentiles) and will be analysed with the independent According to the standard protocol guidelines, the authors declare that unblinded data of the trial will not be presented prior to the release of the main results. Unblinding will occur at the end of the study. A clinical article will be written on the primary and secondary outcomes of the study, and the results will be disseminated regardless of the magnitude or direction of effect. A full study report, an anonymised participant-level dataset and the statistical code for generating the results will be made publicly available no later than 3 years after termination of the study. The present trial is not industry initiated.Clinical experience has suggested that RSB is a very effective postoperative analgesic technique, and we will answer the following questions with this trial.Do preoperative block and postoperative block have the same duration of analgesic effects? Can postoperative block extend the analgesic time? In theory, a single dose of local anaesthetic usually has a maximum duration of 12 hours , and if The results of this study could have actual clinical applications that could help to reduce postoperative pain and shorten hospital stays. In addition, the increasing number of operations performed requires the rapid and accurate delivery of regional block anaesthesia. If good results can be obtained with postoperative block, then we can perform it at the PACU after surgery to speed up the operation flow. To minimise the imbalance of anaesthesiologists and gynaecologists, the same group of gynaecologists will perform the standard surgical procedures, and all anaesthesia will be administered by one anaesthesiologist. The limitation of our study is that we will evaluate only a single-dose RSB because a continuous infusion catheter cannot be used due to the current technical level and higher cost. However, it could be attempted in future studies.Patient recruitment will start in September 2015. The predicted study completion date is December 2016.


Current treatments of various cancers include chemotherapy, radiation, surgery, immunotherapy, and combinations. However, there is a need to develop novel diagnostic and therapeutic treatments for unresponsive patients. These may be achieved by the identification of novel diagnostic and prognostic biomarkers which will help in the stratification of patients' initial responses to particular treatments and circumvent resistance, relapses, metastasis, and death. We have been investigating human prostate cancer as a model tumor. We have identified Yin Yang 1 (YY1), a dysregulated transcription factor, whose overexpression correlated with tumor progression as well as in the regulation of drug resistance and the development of EMT. YY1 expression is upregulated in human prostate cancer cell lines and tissues. We postulated that YY1 may be a potential biomarker in prostate cancer for patients' stratification as well as a novel target for therapeutic intervention. We used Bioinformatic gene RNA array datasets for the expression of YY1 in prostate tumor tissues as compared to normal tissues. Interestingly, variations on the expression levels of YY1 mRNA in prostate cancer were reported by different investigators. This mini review summarizes the current reported studies and Bioinformatic analyses on the role of YY1 in the pathogenesis of prostate cancer. Prostate cancer is a cancer that forms in the tissues of the prostate gland, a tubulo-alveolar exocrine gland that surrounds the male bladder and urethra and is part of the male reproductive system. The primary function of the prostate in the body is to secrete a milky alkaline fluid that is a major constituent of semen. Other non-peptides and prostate-specific antigens are also produced by the prostate and are secreted out. The basicity of secreted fluid helps neutralize the acidity of the vaginal tract and allows for longer survival, better motility, and protection for the sperm .Cancer in the prostate gland occurs usually in men of age 65 or older and once formed, may metastasize from the gland to other parts of the body. Ethnicity and a high fat diet are also very significant risk factors. While morbidity rates have increased significantly, almost two fold from the year 1990 to 2007, there have also been important developments in diagnosing prostate cancer with the help of recent technologies. It. It20]. YY1 gene within cells. Shi et al showed that YY1 was crucial for embryonic development. They used a homozygous mutation in mice which led to lethality and the heterozygous knockout suffered from growth retardation [Reported studies investigated the role of YY1 in metastasis, its role as a therapeutic target, and finally its significance and function as a diagnostic or a prognostic factor ,16. Brieardation . Heterozardation .c-myc, c-fos, EIA, p53, ERB B2 [p53, Mdm2, Ezh2, R6, caspases and HDACs [Noteworthy, recent interest has shifted due to YY1 level of expression and its various functions in different cancers. For example, YY1 has been shown to regulate many genes that are implicated in cancers such as , ERB B2 ,22. In and HDACs ,24.et al [et al [et al [Most reported studies showed that YY1 acts as a transcription factor, an activator or a repressor, through directly binding to promoters or as a cofactor ,22. For et al  reportedet al ,22,26. Sl [et al  and Yangl [et al  reportedB23 gene, a gene which suppresses multiple tumor suppressors and, hence, exhibits oncogenic effects. The repression of such a gene leads to a stronger role of YY1 in liver tumorigenesis [et al have reported that, in general, overexpression of the YY1 protein has a tendency to serve as an activator of tumorigenesis and that it is associated with malignancy of bone tumors [et al showed that HLJ-1 is a tumor suppressor that inhibits tumor angiogenesis and metastasis and that the overexpression of YY1 with another activator protein (AP)-1 causes the initiation of the transcription for HLJ-1 and enhances the transcription by a 3-fold increase [YY1 has contradictory-mediated effects. One example is the interaction between the Hepatitis C virus core and YY1. YY1 recruits a coactivator, p300, and a nucleolar phosphoprotein, B23, to attach it to its binding site and activates gene expression when the HCV core is present. HCV interacts directly with the C-terminal end of B23 and uses it to relieve the repression effect of YY1 on B23. When the core is absent, YY1 recruits histone deacetylase 1(HDAC1) to repress the igenesis . DeNigrie tumors . Deletioe tumors . In thisincrease ,30.Metastasis is the process during which primary tumors break off and invade normal tissues at distant sites leading to the production of secondary tumors. It is important to figure out the location and identification of primary tumors before they attack other locations since most patients are diagnosed with cancer at a time after the cancer had spread . It has The expression and activities of YY1 are reported in both normal and cancerous cells. YY1 may activate one or more genes while deactivating another gene(s) that will result in a different phenotypic effect . Most caet al showed that in hypoxic stress conditions in lung cancer, the inhibition of YY1 reduced the buildup of an important transcription factor, HIF-1α, in a p53-independent manner and that the inhibition of YY1 also resulted in the suppression of the metastatic potential in cancer cells [et al [Wu er cells . HIF-1α er cells . To studs [et al  investigs [et al .HER2 gene and YY1 has been shown to play a critical role as a cofactor in stimulating AP-2 transcriptional activity. Powe et al studied the implications of using YY1 and AP-2 as therapeutic targets in breast cancer and found that AP-2 alpha/beta correlated significantly with YY1 and could be used as important markers for the prognosis of cancer [The human epidermal growth factor receptor 2 (HER2) is a protein that is commonly overexpressed in high grade breast cancer cells and is frequently associated with tumor growth and metastasis ,49. The f cancer .et al [Human meningiomas and astrocytic gliomas are the most common type of cancers found in the central nervous system (CNS) and make up 20% and 35% of all CNS tumor cases, respectively ,52. Mostet al  quantifiet al .et al [et al also found that there is an association between YY1 and E2F, a transcription factor that is critical in the control of the cell cycle and tumor suppressing protein actions [YY1 overexpression in ovarian cancer, in contrast to most cancers, correlated with higher ovarian cancer survival rates . Matsumuet al  studied et al . Unlike et al . Becauseet al ,58. Mats actions .et al [in vitro protein studies and immunoprecipitation, that there is in fact a protein-protein interaction between the AR and YY1. The AR regulates prostate development and its interaction with YY1 optimizes its transcriptional activity. They observed that a normal amount of YY1 within prostate cells does in fact optimize its transcriptional activity but overexpression of YY1 causes it to lose its function. When YY1 is overexpressed, the excessive YY1 interacts with the AR and other cofactors which then interfere with the formation of a transcription complex. Consequently, not only does this keep the complex from optimal transcriptional activity but causes an inverse effect on the expression of AR-targeted genes due to the squelching effect of excessive amount of YY1 present [Having a certain level of YY1 expression is considered normal within certain cells and is necessary for the YY1 to maintain its function. Deng et al  studied  present ,59.et al [Androgens interaction with the AR on prostate cancer cells stimulate proliferation and progression . The traet al  studied PSCA is an androgen-responsive gene through the interaction of AR with AREs [et al [in vivo, YY1 interacts with the PSCA protein as assessed by chromatin immuno-precipitation assay (3) much of YY1 binding sites demonstrated that YY1 has a dual opposing control of PSCA activation; activation of both binding sites generated intermediate luciferase activity (4) YY1 knockdown induces androgen-mediated upregulation of PSCA and (5) YY1 siRNA in PC3 (AR-mutated) increased endogenous PSCA expression.PSCA is a GP1-anchored cell surface protein  and is aith AREs . Other rith AREs . YY1 alss [et al  who repoet al [et al [Seligson et al  were thel [et al  investigl [et al .et al [Sun et al  carried et al . These fTreatment of various cancers includes surgery, chemotherapy, radiation, immunotherapy and combinations thereof. These various modalities result in significant clinical responses and seldom cures. However, an initial subset of cancer patients remains unresponsive and another responding subset develops resistance and often cross-resistance to different therapeutic modalities. Clearly, these subsets of patients require novel treatments that can overcome resistance. Approaches that are currently being investigated consist of targeting resistance factors directly at the tumor cells or indirectly at the tumor microenvironment. Targeting resistance factors include agents that interfere with constitutively activated survival/anti-apoptotic pathways that usually control resistance. Our laboratory has examined a gene product, namely, YY1, that is overexpressed in many cancers including prostate cancer. We have reported that YY1 is involved in the regulation of both drug and immune resistance.We have reported that tumor cells that are resistant to FasL-mediated apoptosis can be sensitized following treatment with interferon-gamma (IFN-γ). One of the many downstream targets of IFN-γ is the enzyme iNOS and, therefore, we examined the role of iNOS in the regulation of tumor cell resistance to FasL. The findings demonstrated that the induction of iNOS following treatment of tumor cells with IFN-γ or treatment with NO donors sensitized the cells to FasL apoptosis. The sensitization was correlated with upregulation of Fas on the cell surface membrane of treated tumor cells. Analysis of the Fas promoter revealed many putative transcription factor-binding sites including for YY1. We established the role of YY1 in the resistance by demonstrating that it is inhibited by IFN-γ, iNOS and NO donors and, also, YY1 represses the transcription of Fas. In addition, treatment of tumor cells with siRNA YY1 resulted in the up regulation of Fas and sensitization to FasL apoptosis . These fet al [et al. and demonstrated that treatment of DU145 human prostate carcinoma cells with the NO donor DETANONOate sensitized the drug resistant tumor cells to apoptosis to various chemotherapeutic agents [in vitro findings were corroborated in vivo in mice bearing a PC3 tumor xenograft and treated by the combination of DETANONOate and CDDP [Wink et al  demonstret al . The nitet al . Our stuc agents . The senand CDDP .Altogether, the above findings demonstrated clearly that YY1 plays a direct role in the regulation of tumor cells resistance to both chemotherapeutic drugs and immune-mediated apoptosis. These findings suggest the potential therapeutic application of YY1 inhibition in the reversal of patient tumors refractory to conventional treatments.Many solid cancers, in general, develop molecular, biochemical, genetic and epigenetic mechanisms to evade the response to the host immune surveillance and external cytotoxic activities . These fA study of YY1 expression levels in various carcinomas in the Oncomine Bioinformatics Database reaffirms the predicament researcher's face in giving YY1 a good or bad label. While high YY1 expressions in lung, breast, and brain and CNS cancers correspond to a high likelihood of cancer metastasis, high expressions of the same YY1 in ovarian cancer increases survival rates .Oncomine Bioinformatics Database is a cancer microarray database that is useful to perform differential analyses of gene expression using data from hundreds to thousands of patients. It permits researchers to get a comprehensive idea of gene expression by searching the database for specific mRNA levels of genes in normal tissues versus cancerous tissues in various kinds of cancers. Researchers upload their data from their experiments after their publication for the general public. Of importance, researchers could use these data to their full potential and can demonstrate trends in gene expression levels in different cancer types and use the data to look for diagnostic or prognostic markers and potential therapeutic targets since all of the data are available with publication references, supplementary data, and annotations .Because the above data are uploaded at different times and the samples used have been exposed to different experimental methods, the data are expected not to be uniform and may be dissimilar. What is clear is that each dataset may have been interpreted by the investigators in a certain way but clearly many more alternative interpretations may derive when all the datasets are examined concurrently.et al [et al [We have analyzed the YY1 mRNA expression levels using available public datasets of microarrays that were retrieved from the Oncomine Database and gene expression Omnibos, derived from the published analyses reported by several investigators -83. The et al  and Ambsin vitro by using preclinical animal models bearing human tumor xenografts. We also suggest that it is important to find out whether the NF-κB/Snail/YY1/RKIP/PTEN dysregulated circuitry found in the EMT phenotype is also shared in cancer stem cells such that YY1 inhibitors will be helpful to prevent the development of a metastatic tumor from stem cells and induce the death of stem cells [This review has briefly discussed the role of YY1 in the pathogenesis of prostate cancer as a model. Clearly, overexpression of YY1 has been noted in many cancers and was shown to play a pivotal role in the regulation of EMT and metastasis as well as its role in the regulation in tumor cell resistance to both chemotherapy and immunotherapy. Therefore, YY1 is proposed to be a therapeutic target for intervention as well as a potential prognostic biomarker in prostate cancer. There are several agents that inhibit YY1 expression such as proteasome inhibitors , nitric em cells . We also


HMOX1 SNPs rs2071746, rs2071747, and rs9282702, a microsatellite (GT)n polymorphism and copy number variations in 691 patients suffering from PD and 766 healthy control individuals. Copy number variations in the HMOX1 gene exist, but these do not seem to be associated with PD risk. In contrast two polymorphisms that modify the transcriptional activity of the gene, namely a VNTR (GT)n and the SNP rs2071746, are strongly associated with PD risk, particularly with the classic PD phenotype and with early onset of the disease. This study indicates that HMOX1 gene variants are associated to the risk of developing some forms of PD, thus adding new information that supports association of HMOX gene variations with PD risk.The blood-brain barrier (BBB) supplies brain tissues with nutrients, filters harmful compounds from the brain back to the bloodstream, and plays a key role in iron homeostasis in the human brain. Disruptions of the BBB are associated with several neurodegenerative conditions including Parkinson's disease (PD). Oxidative stress, iron deposition and mitochondrial impaired function are considered as risk factors for degeneration of the central nervous system. Heme oxygenase (HMOX) degrades heme ring to biliverdin, free ferrous iron and carbon monoxide being the rate-limiting activity in heme catabolism. The isoform HMOX1 is highly inducible in response to reactive oxygen species, which induce an increase in BBB permeability and impair its pathophysiology. Consequently, an over- expression of this enzyme may contribute to the marked iron deposition found in PD. We analyzed the  The blood-brain barrier (BBB) supplies brain tissues with nutrients, filters harmful compounds from the brain back to the bloodstream, and plays a key role in iron homeostasis in human brain. Disruptions of the BBB are associated with several neurodegenerative diseases including Parkinson's disease (PD). Oxidative stress, iron deposition and mitochondrial impaired function are considered as risk factors for degeneration of the central nervous system  enzyme activity degrades heme ring to biliverdin, free ferrous iron and carbon monoxide being the rate-limiting activity in heme catabolism . In addition, 231 patients with PD were recruited from the Hospital de La Princesa  and 307 controls were included from the latter hospital and from the Infanta Cristina University Hospital . Most participants had been included in previous studies by our group . These polymorphisms included a variable number tandem repeat (VNTR), consisting of alternating the purine-pyrimidine sequence (GT)n which has the potential to acquire the Z-DNA conformation, a structure which is thermodynamically unfavorable compared with the B-DNA structure. This formation has been described as a factor that negatively affects transcriptional activity  and class L including long repeat sequences (n ≥ 25)  located at 22:35386857, which corresponds to exon 3 of the HMOX1 gene. Besides these SNPs, no other non-synonymous HMOX1 SNPs have been reported to occur in Caucasian individuals at frequencies over 1%. Custom primers were designed to analyze the SNPs rs9282702 and rs2071747, whereas commercial primers were used for the detection of the SNPs rs2071746 . The detection was carried out by qPCR in an Eppendorf Realplex thermocycler by using fluorescent probes. The amplification conditions were as follows: After a denaturation time of 10 min at 96°C, 45 cycles of 92°C 15 s 60°C 90 s were carried out and fluorescence was measured at the end of each cycle and at endpoint. All samples were determined in triplicate and genotypes were assigned both by gene identification software  and by analysis of the reference cycle number for each fluorescence curve, calculated by the CalQPlex algorithm (Eppendorf). For technical validation purposes, the amplified fragments for 20 individuals carrying every genotype : The SNP rs2071746 (A/T) is an upstream variant located at 22:35380679, rs2071747 is a missense mutation G/C (Asp/His) located at 22: 35381192, which corresponds to exon 1 of the HMOX1 gene were analyzed using the TaqMan copy number assay Hs00774483_cn (Applied Biosciences Hispania), which was designed to hybridize within the open reading frame in HMOX1 gene exon 3 (chromosome location 22:35782935). Amplification was carried out in an Applied Biosystems 7500 real-time thermocycler as described by the manufacturer, using as a copy number reference assay RNAse P. All reactions were carried out in quadruplicate. Results were analyzed by means of the CopyCaller Software (Applied Biosciences Hispania). According to standard procedures in CNV analyses, we designed as heterozygous (null/present) those samples with a single copy of the HMOX1 gene.Copy number variations (CNVs) of the http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Allelic and genotype analyses were performed with the PLINK software  procedure as described elsewhere . The statistical power for two-tailed associations for the presence of any of the two SNPs identified in this study was 99.9% for overall patients with PD, 98.3% for patients with C-PD and 95.4% for patients with an unknown PD phenotype. Testing for heterogeneous association (homogeneity test) was analyzed by using the Breslow–Day test.The Hardy–Weinberg equilibrium was analyzed with the DeFinetti program . The best fit was observed with the dominant and the allelic models. We identified a statistically significant difference of the rs2071746 genotypes between patients with C-PD and healthy individuals. This difference was observed both in the dominant model and in the allelic model. After correction for the three PD presentations , these associations remained statistically significant in the C-PD group. In addition, a weak statistically significant difference in the frequency of HMOX1 rs2071746 genotypes between patients with TD-PD and healthy individuals was observed. This difference followed a dominant model, but the difference was not statistically significant in the allelic model , and no particular risk haplotypes were identified in any of the study groups.The combined genotypes obtained after SNP and CNV analyses are summarized in Table HMOX1 rs2071746 genotype frequencies were related to age of onset in the C-PD subgroup. Patients with C-PD phenotype were subdivided according to the median age at onset in this subgroup (62 years). Table As has been described previously, patients with the phenotypes TP-PD and C-PD may have an earlier age at the onset than the AR-PD group  repeats in healthy subjects and GT(22) repeats in the case of PD patients. Mann–Whitney comparison for means in each group revealed statistically significant differences (p < 0.001). The association study of this polymorphism with PD patients is shown Table Regarding the HMOX-1 GTHMOX1 mRNA upregulation in CNS is a marker of oxidative stress. The enhanced expression of HMOX1 may contribute to the pathological deposition of iron which has been reported in normal aging CNS, and to a much greater extent in neurodegenerative disorders such as PD  with C-PD. The rs2071746 A allele was more common among PD patients with C-PD phenotype than in control individuals. Also, we observed that this association was related with an early onset of the disease. In addition, the analysis of the length of the highly polymorphic (GT)n dinucleotide repeats indicates an association of longer (GT)n repeats with susceptibility to PD. Both gene variations, rs2071746 and (GT)n dinucleotide repeats sequence, have been related to variations in the transcriptional activity of the HMOX1 gene  among PD patients and an increased frequency of carriers of the rs2071746 A allele among PD patients. Although these findings apparently point to opposite directions in terms of gene activity, the functional effect of these polymorphisms is not fully understood. Moreover, an extended model of the HMOX1 gene along with the importance of alternative splicing in the area where these two polymorphisms are located, and its association with translational regulation has been disclosed recently (Kramer et al., We identified a clinical association of the HMOX1, both in PD patients and in healthy individuals. We found CNVs more frequently in AR-PD patients. Although this study does not support a major role of HMOX1 CNVs in PD risk, the demonstration of the occurrence of CNVs in the HMOX1 gene supports further research in the functional role and clinical associations of HMOX1 CNVs. It is of note, however, that the association cannot be observed in the subgroup of PD patients with unknown phenotype. Our findings suggest that the association of HMOX1 genotypes and PD risk is specific of the C-PD phenotype. This reinforces the need of detailed phenotyping and categorization of patients in genetic association studies, particularly when assessing low-penetrance factors. In summary, in this study we identified two functional HMOX1 gene variations as potential genetic biomarkers of PD. It is conceivable that these genetic biomarkers would be associated to other disorders related to oxidative stress in the CNS and in the BBB. This study adds to current knowledge of genetic biomarkers in PD (Alonso-Navarro et al., HMOX1 genetic variations in an independent population to verify these associations and to analyze putative interethnic variability in the observed genetic association.Our results agree with those by Kimpara et al.  althoughThe 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.


Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from  Saccharomyces cerevisiae these are Cdc42, Rac , Rho1, Rho2, Rho3 and Rho4. While some fungi contain additional Rho paralogs that seem to have been acquired by horizontal gene transfer (e.g. Aspergillus fumigatus and Neurospora crassa), some species within the above named groups carry additional copies which most probably stem from duplication events. This is, for example, the case for Rho1 in Ashbya gossypii (see below) and Schizzosaccharomyces pombeS. cerevisiae. For a long time it was generally accepted that an Rac ortholog is not present in this yeast, but some functional analysis data, and more recent phylogenetic analyses, suggest that Rho5 might be a true Rac-ortholog in S. cerevisiaeSchizzosaccharomyces pombe seems to be the only fungal model organism that does not possess a Rac-homolog The family of small GTP-binding proteins of the Rho-type is composed of important regulators of the actin cytoskeleton in nearly all eukaryotic organisms. Because Rho-homologs are highly redundant in metazoan cells, the analysis of specific functions of single proteins is complicated. Therefore, model organisms with less Rho-GTPase redundancy are valuable tools for deciphering Rho-GTPase function and evolution. Often fungi are used for this purpose. A recent survey that is based on datasets containing many complete genome sequences suggests that generally six groups of paralogs exist in fungi N. crassa , Aspergillus nidulans Ustilago maydis Ashbya gossypiiSchizzosaccharomyces pombeS. cerevisiae, simply because the earliest attempts at studying Rho-proteins in a fungus were made in this organism S. cerevisiae. Therefore, the function of many members of the Rho-family in S. cerevisiae is very well understood. This is the case for Cdc42, Rho1, Rho3 and Rho4. They play important roles as far as cell polarity, cytokinesis, cellular integrity and secretion are concerned, and, accordingly, mutants of these genes present clear phenotypes. However, the function of the other members of this protein family is less well characterized, often simply because no clear phenotype is associated with mutants of the corresponding genes.Analysis of Rho-GTPase function has been carried out in many fungal model organisms including Saccharomyces cerevisiae had a more severe phenotype in the close-relative species Ashbya gossypiiA. gossypii often provide novel and additional information on gene function. All family members of the Rho-type GTPases from S. cerevisiae are also conserved in A. gossypii. Currently, AgCDC42, AgRHO3, AgRHO1a and AgRHO1b have been characterized in A. gossypiiAgCdc42 is essential for the switch from isotrophic to filamentous growth during the germination of the spore and, most likely, for polarity at the hyphal tip, where the protein localizes Agcdc42 fail to leave the isotropic growth phase after germination and consequently die RHO1 gene has two homologs in A. gossypii. This duplication is not unique to A. gossypii, but is rather conserved in several species of the Saccharomycetaceae, including the putative ancestor of S. cerevisiae and A. gossypii that has been calculated from the genomes of several yeast species Agrho1b show phenotypes that are very similar to Scrho1 mutants and suggest a function in cell wall biosynthesis and cellular integrity RHO1 gene, AgRHO1a, which is present in the genome of A. gossypii, plays an important role in maintaining cellular integrity under high hyphal growth speeds A. gossypii RHO1a gene carries a unique mutation that is most likely associated with the filamentous nature of A. gossypii and allows for a decoupling of AgRho1a and AgRho1b activation We have previously shown that some mutations of syntenic homologs to the genes of RHO2 and RHO5 genes are less understood. RHO2 and RHO5 have been only poorly characterized in S. cerevisiae. Rho2 is thought to have a partially overlapping function with Rho1 in actin cytoskeleton repolarization In contrast to the genes described above, the functions of the Agrho2 and Agrho5 mutants in Ashbya gossypii. We show that both of the GTPases have strong phenotypes that affect hyphal growth and morphology. Additionally, we demonstrate that the Agrho5 phenotype is copied by a mutant in a putative Agrho5 GDP-GTP exchange factor (GEF). The latter gene encodes a homolog to members of the DOCK-homology family, which are hypothesized to act as putative GEFs for Rac homologs in other fungal organisms Here, we report on the phenotypes of Ashbya gossypii strains was performed using PCR-based gene targeting, as described by Wendland et al.A. gossypii with plasmids was performed according to the protocol by Wright et al.Agrho2*-strain, the ScLEU2 gene was integrated on a vector behind the G195CAgrho2 allele ScLEU2-marker and the flanking sequences and was transformed into the Agrho2 deletion to replace the GEN3 deletion marker.The construction of all A. gossypii was cultured in Ashbya Full Medium  E. coli and S. cerevisiae were isolated using the High Pure Plasmid Isolation Kit . For plasmid isolation from yeast, we used a modified protocol by Schmitz et al.DNA manipulations were performed as described previously et al.For fluorescence microscopy, we used the protocol described in Kemper ACL087C (AgRHO2), ABL139C (AgRHO5), AER102W (AgDCK1) and ACR130W (AgABP140).The GenBank IDs for the genes are: AgRho2 protein, we first constructed a deletion mutant by replacing the entire open-reading frame of the encoding gene by PCR-mediated gene targeting with a marker cassette. In addition, we made use of the fact that the GTPase structure and its regulation are highly conserved among Rho-type GTPases. This enabled the construction of GTP-locked mutants by the simple mutation of a conserved glutamine in the switch II region. We constructed such a mutant for AgRHO2 (Agrho2*) in vitro and integrated it at the original AgRHO2 locus in the genome. rho2 mutants. While the deletion mutant grew slightly but significantly faster than the wild type, the Agrho2* mutant consistently grew slower. Interestingly a simple overexpression of the wild-type allele did not yield the same result, but grew rather like the wild type  Dck1 is required for Rac1 activation during invasive filamentous growth. We identified a homolog to DCK1, AER102W, in the genome of A. gossypii and speculated that this might be a potential regulator for AgRho5 in A. gossypii. To test this hypothesis, we also deleted the gene AER102W by direct gene targeting. As shown in Agrho5 deletion. In addition, both deletion strains were more sensitive to the actin destabilizing drug Latrunculin A than the wild type. The similarities between the two deletion mutants became even more evident when comparing the microscopic growth defects. A. gossypii, after an initial isotropic growth phase, a germ-tube is formed at an angle that is close to 90° to the spore needle axis. We determined the deviation of the first germ-tube for the wild type and for both deletion mutants. This deviation was only 14.6° for the wild type, while it was 25° for the Agrho5 and 22.4° for the dck1 deletion. This polarity defect was maintained during formation of the second germ-tube  along with the observed similar phenotypes, suggests that, in vegetatively growing cells, AgPxl1 could be involved in the regulation of AgRho2 at the hyphal tip. This is in agreement with the fact that a GFP-fusion of AgPxl1 localized to the hyphal tip and to septa in vegetatively growing hyphae. However, our attempts to prove such an interaction by two-hybrid experiments and by co-immunoprecipitation with purified proteins were both unsuccessful. Further work will be required to ascertain whether there is either no connection, or only an indirect link between the highly similar defects of Agrho2 and Agpxl1 mutants.A similar, asynchronous tip-branching defect which we observed for the AgRho2 appears to not play a role in spore formation, although its GFP-fusion localized to sporangia. Spore formation was normal, and we could not observe an effect on spore length as we did for the mutants of Agrho1 or Agrho1b. Therefore, AgRho2 appears to have a regulatory role at the hyphal tip that is essential for proper tip-branching. Together with other tip-branching mutants, such as the Agpxl1 deletion AgBni1, that display premature tip-branching Agrho2 mutant provides further evidence that tip-branching is a highly regulated process.Interestingly, AgRho2 appears to be required for branching, mainly at the tip at high-growth speeds, AgRho5 is required early in the development of A. gossypii. The Agrho5 deletion dramatically affected growth speed but not polarity. The hyphae appeared to be perfectly pointed and only the direction of polarity changed. These polarity changes already started at the germinating spores. The deletion of AgDCK1 was an exact phenocopy of the Agrho5 deletion, which suggests that both deletions function as activators of the same processes and supports the idea that RHO5 homologs are divergent Rac-homologs AgDCK1 is a homolog of the DOCK-family of proteins which, together with proteins of the so-called ELMO family, form a novel class of GTP-GDP exchange factors (GEFs). Even though direct biochemical proof still has to be provided, there is strong evidence to support the conclusion that the homolog of AgDCK1 in C. albicans functions as a putative GEF for the CaRac1 protein A. gossypi and Rho5 homologs are absent in all filamentous fungi that possess a Rac homolog AgRho5 protein has acquired some of the functions of the Rac protein and that the AgDck1 protein is the evolutionarily conserved GEF that regulates these functions. The processes that appear to be regulated by AgRho5 further support this hypothesis. Rac homologs in most fungi are responsible for hyphal growth, polarity maintenance and actin organization and have a varying degree of importance for the regulation of these processes S. cerevisiae, Rho5 was shown to bind to Ste50 and to be involved in the regulation of the osmotic stress response A. gossypii rho5 deletion mutant. In contrast to S. cerevisiae, where Rho5 is involved in oxidative stress response AgRho5 in response to oxidative stress (data not shown).While Agrho5 and Agdck1 mutants, the instability of the actin structures at the hyphal tip and at septation sites and the sensitivity to Latrunculin A, may also point to proteins that may be involved in the affected processes. The homolog to the AgDck1 protein of S. cerevisiae physically interacts with Lsb3 (Las seventeen binding), which, in turn, binds to Las17 LAS17 homolog WAL1 (AgLAS17) in A. gossypii completely lack actin rings Agrho5 and Agrho2 is lethal suggests further that both proteins signal into an additional branch independently, which is distinct from the signaling branch that is linked to the phenotype observed in the single deletion mutants. In addition, our result with an Agrho2/Agrho5 double deletion suggests that the Agrho5 deletion phenotype is dominant over the Agrho2 deletion phenotype. Given the fact that both phenotypes are related to hyphal growth, this is not surprising, as the effect of the Agrho5 deletion severely impairs hyphal growth, whereas the effects of an Agrho2 deletion are much less pronounced.A characteristic feature of the phenotype of the S. cerevisiae only cause mild defects - emphasize the value of A. gossypii as a complementary, morphologically more complex model system to the yeast cells of S. cerevisiae.The strong phenotypes for the homologs of two Rho-family members that we have identified in this study - the mutants of which in Movie S1Supporting movie for , showing the complete information that was used to generate the data shown in .(AVI)Click here for additional data file.Movie S2Supporting movie for , showing the complete process that was analysed to generate .(AVI)Click here for additional data file.


If the photoconversion in the skin due to reduced sun exposure  is insufficient, intake of adequate vitamin D from the diet is essential to health. Severe vitamin D deficiency can lead to a multitude of avoidable illnesses; among them are well-known bone diseases like osteoporosis, a number of autoimmune diseases, many different cancers, and some cardiovascular diseases like hypertension are being discussed. Vitamin D is found naturally in only very few foods. Foods containing vitamin D include some fatty fish, fish liver oils, and eggs from hens that have been fed vitamin D and some fortified foods in countries with respective regulations. Based on geographic location or food availability adequate vitamin D intake might not be sufficient on a global scale. The International Osteoporosis Foundation (IOF) has collected the 25-hydroxy-vitamin D plasma levels in populations of different countries using published data and developed a global vitamin D map. This map illustrates the parts of the world, where vitamin D did not reach adequate 25-hydroxyvitamin D plasma levels: 6.7% of the papers report 25-hydroxyvitamin D plasma levels below 25 nmol/L, which indicates vitamin D deficiency, 37.3% are below 50 nmol/Land only 11.9% found 25-hydroxyvitamin D plasma levels above 75 nmol/L target as suggested by vitamin D experts. The vitamin D map is adding further evidence to the vitamin D insufficiency pandemic debate, which is also an issue in the developed world. Besides malnutrition, a condition where the diet does not match to provide the adequate levels of nutrients including micronutrients for growth and maintenance, we obviously have a situation where enough nutrients were consumed, but lacked to reach sufficient vitamin D micronutrient levels. The latter situation is known as hidden hunger. The inadequate vitamin D status impacts on health care costs, which in turn could result in significant savings, if corrected. Since little is known about the effects on the molecular level that accompany the pandemic like epigenetic imprinting, the insufficiency-triggered gene regulations or the genetic background influence on the body to maintain metabolic resilience, future research will be needed. The nutrition community is highly interested in the molecular mechanism that underlies the vitamin D insufficiency caused effect. In recent years, novel large scale technologies have become available that allow the simultaneous acquisition of transcriptome, epigenome, proteome, or metabolome data in cells of organs. These important methods are now used for nutritional approaches summarized in emerging scientific fields of nutrigenomics, nutrigenetics, or nutriepigenetics. It is believed that with the help of these novel concepts further understanding can be generated to develop future sustainable nutrition solutions to safeguard nutrition security.Vitamin D is a micronutrient that is needed for optimal health throughout the whole life. Vitamin D Vitamin D is needed to maintain calcium concentrations within a narrow physiological range. This function is vital as the calcium ion is essential for a large variety of cellular and metabolic processes in the body  and ergocalciferol (vitamin D2), whereas the metabolite 25-hydroxycholecalciferol (25-hydroxyvitamin D3) is a natural part of the food chain by its occurrence in animal products. Vitamin D3 is unique by the fact that the same nutrient can be synthesized in the skin through the action of sunlight or being taken up by diet. This dual source of intake secures the body to maintain sufficient vitamin D levels in the body. The production in skin is usually the major vitamin D3 source for the body. However, in countries that receive insufficient sun exposure, people rely on dietary vitamin D as a major source. Exposure of the precursor 7-dehydrocholesterol in the basal and suprabasal layers of the epidermis to ultraviolet B (UVB) light with a wavelength of 290–315 nm is needed for the formation of the previtamin D3. The subsequent conversion is a non-enzymatic process that includes a thermal isomerization of the previtamin D3 to produce vitamin D3  or dietary contribution (vitamin D3 and vitamin D2). The 25-hydroxyvitamin D3 needs to be further hydroxylated in the kidney . The plant-derived vitamin D2 is processed in the same way. For both vitamers, vitamin D2, and vitamin D3, the consecutive molecular action is believed to be identical, whereas only 1,25-dihydroxy vitamin D3 is the endogenous hormone, the activated vitamer 1,25-dihydroxyvitamin D2 is hormone mimetic. Therefore, it was not surprising that vitamin D3 has been reported to be superior to vitamin D2 in terms of bioavailability and maintaining the vitamin D status by the majority of studies . Therefore, dietary intake of vitamin D through foods or supplements plays a vital part to maintain healthy vitamin D levels.The level of cutaneous vitamin D2 that is only present in some mushrooms. Commercially dark cultivated white button mushrooms contain low amounts of vitamin D2, only wild mushrooms or sun-dried mushrooms contain elevated amounts of ergocalciferol , contain a glycoside of the active 1,25-dihydroxyvitamin D3 hormone  and margarine (13.3 μg/100 g) , which represents the safe upper limit, was set to 4000 IU per day for vitamin D intake  introduced new dietary reference intake (DRI) values for vitamin D after comprehensive reviewing of more than 1000 high quality research articles to renew thereby their first settings from 1997  and FIND study , are meant to deliver clinical evidence for the effect of vitamin D3 on cancer, CVD and diabetes outcomes. The two smaller trials, CAPS and DO-HEALTH, each having more than 2,000 participants are including cancer, infections, fractures, hypertension, cognitive function, and physical performance outcomes. In all four studies the placebo group will produce vitamin D3 in the skin and will possibly consume vitamin D through food, and therefore this will narrow the vitamin D serum level gap between the placebo and treatment groups. It remains to be seen whether the applied supplementation doses  will be sufficient to see a clear difference between the treatment and the control groups. An open likelihood will remain for the placebo group potentially obtaining sufficient vitamin D3 (600–800 IU) levels that are considered to be sufficient for skeletal effects. In such a case only an incremental increase of an additional ~1000 IU can be considered as the effective dose, for which no power calculation was available at the time before study begun. In light of such a situation, it will be of interest whether the micronutrient triage theory of Bruce Ames can be validated with vitamin D3  and gets transported into the liver , in the regulatory region of primary 1,25-dihydroxyvitamin D3 target genes  or histone deacetylases (HDACs) and epigenically direct transcriptional expression of downstream genes . A number of other countries around the globe are in the process of establishing new dietary intake recommendations as well. It turns out that on average a daily intake of 600–800 IU vitamin D appears to be required to meet fundamental needs of the human body, for specific applications higher daily intakes may be necessary, which will become clearer as the results of a number of ongoing clinical studies will become available.The obvious question to answer is: do people obtain the recommended amounts of vitamin D? The diet is typically only a minor vitamin D source as only few food items contain relevant amounts of vitamin D, such as fatty sea fish. The primary vitamin D source for humans is the vitamin D synthesis in the skin from vitamin D precursors by the sunlight—provided the skin is sufficiently exposed to strong enough sun radiation. Several groups have reviewed the published results on 25-hydroxyvitamin D serum levels the established marker of the vitamin D status, showing that low 25-hydroxyvitamin D levels are found in many cohorts around the world. A recent systematic review of the global vitamin D status (Hilger et al., In addition, it will be very important to gather sound and convincing evidence for the many additional implicated health benefits of vitamin D besides the ones that already reached a health claim status and to see which of them will actually hold up. This will require appropriate human studies on the one hand, and also involve the appropriate use of the novel experimental approaches like nutrigenomics, nutrigenetics, and nutriepigenetics on the other hand. In conclusion, the evidence we have for vitamin D in human health is exciting, however we have to make sure that appropriate measures are taken to improve the vitamin D status to the levels required to be beneficial for human health. In future, we will also need to further apply, exploit and invest in novel, innovative and break-through technologies in the vitamin D research to understand the underlying mechanisms by which vitamin D is exerting so many effects in the human body, which is knowledge needed to the purpose to obtain and secure optimal public health through nutrition.The authors are employees of DSM Nutritional Products Ltd. and declare to have no conflict of interest.


We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson–Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that,  RecA family proteins play important roles in DNA recombination and repair. The proteins include RecA itself, which promotes strand exchange in bacteria, as well as Rad51, which promotes strand exchange in eukaryotes. At the beginning of a repair or recombination process, a region of single-stranded DNA (ssDNA) is formed at a DNA double-strand break. The single-stranded region, referred to as the initiating strand, is covered by a recombinase protein forming a helical filament with 6 RecA molecules per helical turn . The binIt is believed that during homology testing, the dsDNA binds to the secondary binding site in RecA –10. AfteThe crystallographic structure of the dsDNA bound to site I, referred to as the RecA-heteroduplex complex, is well known . It cons−5. Thus, experimental results presented in this work indicate that the vast majority of 20-nt tests that would occur in vivo would not result in the formation of a detectable strand exchange product, suggesting that those interactions are very rapidly rejected. Such swift rejection of almost all interactions in vivo could allow RecA-mediated homology recognition to rapidly search a bacterial genome even if some rare sequences required longer to resolve . Complete sequences of the oligonucleotides used in this work are shown in Supplementary Tables S1 and S2. Lambda phage DNA samples used for single-molecule experiments were prepared by annealing and ligating biotinylated oligonucleotides.FRET experiments for ssDNA binding to the filament were performed by adding a small aliquot of ssDNA to the filament solution in a quartz cuvette. For ssDNA annealing, a small volume of the rhodamine-labeled sequence (Rho c) was added to each fluorescently labeled sequence. FRET strand exchange experiments were performed by mixing a solution containing the unlabeled filament preparation incubated at 37°C for 10 min in the cuvette with a small aliquot of labeled dsDNA. The detection of the emission of the fluorescein label was verified by using 493-nm excitation during 30 min, and the emission was read at 518 nm. The integration was 0.5 s and the band width 2 nm. The sample was kept at all times at 37°C.2 and 5 mM dithiothreitol, pH 7.6). Labeled 20-bp dsDNA was prepared from the annealing of the labeled complementary oligonucleotides by cooling down from 80 to 40°C with 2°C steps equilibrated for 1 min; the emission between 500 and 560 nm was acquired (excitation at 493 nm) at each temperature step. The final concentration of dsDNA once added to the filament preparation was 12 μM in bases.The filament was obtained by mixing each reagent to yield a final preparation of 6 μM in bases of the corresponding oligonucleotide, 2 μM of RecA protein (New England Biolabs), 1 mM ATPγS (Sigma-Aldrich), 0.1 μM SSB (single-stranded binding protein Epicentre) and RecA buffer , and another with 15 mismatches (pcDNA3). In addition, repetitions performed throughout several days were also analyzed. In the former case the intra-assay accuracy showed a variation between 2 and 4% whereas the inter-assay variation was 6–8% in all of the cases. Binding of ssDNA oligonucleotides to filaments (site I) and annealing experiments were repeated at least twice for all of the cases yielding a 2% variation.In single-molecule experiments, we used 3′3′ and 3′5′-biotin labeled dsDNA to attach to extravidin molecules adsorbed on a glass surface and superparamagnetic beads . After an aliquot of dsDNA in RecA buffer was mixed with RecA (New England Biolabs), ATPγS, and the beads, the resulting sample was placed in a square micro-cell with cross-section of 0.8 mm containing a round inner capillary, 0.55 mm in diameter and closed at its ends. The inner capillary was modified by adsorption of 1 mg/ml extravidin (Sigma) in PBS  pH 7.4 overnight at room temperature. After an initial incubation of 4 min, the DNA molecules became tethered between the glass capillary surface and the extravidin-coated beads. The micro-cell was then placed in a magnetic tweezers apparatus consisting of a stack of permanent magnets held at a variable distance from the sample. The 5–200 pN force on the beads is controlled by the distance between the magnets and the micro-cell. The position of each bead is tracked by bead tracking software and recorded as described in previous work .The calculation was done by using the APBS package ,17. HydrFRET based measurements of the sequence dependence of binding of ssDNA to the presynaptic filament, annealing of B-form DNA, and strand exchange.In order to test the proposal that homology recognition originates in the instability of base pairing of mismatched base pairs in the heteroduplex, we performed Förster resonance energy transfer (FRET) experiments to monitor the formation of a RecA-heteroduplex without strand exchange.As earlier work showed, dsDNA in the RecA-heteroduplex complex can be obtained without strand exchange using either of two methods: (i) direct dsDNA binding to site I, or (ii) adding complementary ssDNA to the ssDNA-RecA presynaptic filament in which the initiating ssDNA strand is bound to site I  Figure . For theIt is possible that the binding of free ssDNA to the presynaptic filament could be insensitive to mismatches between the free ssDNA and the initiating strand. For example, the free ssDNA could bind to the secondary binding site without interacting with the initiating strand. If the complementary strand binding to the presynaptic filament were insensitive to mismatches, then the fluorescence signal for the heterologous, homologous and nearly matched ssDNA molecules would all be the same; however, the signals for all of these are markedly different indicating that the binding is sensitive to mismatches.in vivo where ∼20% of the interactions would contain 5/20 accidental matches. Sequences containing 16 or 17 matched bases show 40 to 60% fluorescence change with respect to the homologous sequence, suggesting that base pairing in the RecA-heteroduplex complex can be quite stable in the presence of three or four mismatches.Though the binding is sensitive to mismatches, it is in general not very stringent. If homology stringency were perfect, the asymptotic fluorescence value should not change when the ssDNA added to the active filament contains mismatches. Instead a small change in fluorescence was detected when the presynaptic filament interacted with an ssDNA sequence that contains no matched bases when aligned in registration with the initiating strand Figure . The smaThough sequences containing a small number of mismatches can show fairly stable binding, the data clearly indicate that heteroduplexes containing mismatches yield lower equilibrium binding values than the homologous sequence. We note that previous fluorescence studies have also found that the binding of ssDNA in the heteroduplex favors the homologous interaction .Having evaluated the mismatch-dependent instability provided by the binding of free ssDNA to ssDNA bound to site I, we wanted to compare those results to the mismatch-dependent instability provided by the annealing of free ssDNA to form B-form dsDNA under the same conditions. Thus, we hybridized the same fluorescein and rhodamine labeled ssDNA molecules in the absence of RecA protein, as illustrated in Figure In order to study the relationship between the homology stringency provided by the binding of the complementary strand to the presynaptic filament and the stringency provided by strand exchange, we performed additional FRET experiments Figure . These eFigure Figure In Figure The asymptotic values of the fluorescence curves for direct ssDNA binding to an ssDNA-RecA filament indicate that base pairing in the heteroduplex is less stringent than the pairing in B-form dsDNA; however, it does not provide information about the relative stability of the base pairing in the two structures. Crystallographic studies have shown that backbone interactions do not play a significant role in binding the complementary strand to the heteroduplex , so the For these experiments, we formed the RecA-heteroduplex complex by directly binding dsDNA to site I. Single molecules are selected by following the overstretching curve initially when there is no significant amount of RecA protein bound to dsDNA. This curve should show that the overstretching transition takes place at around 65 pN; higher forces indicate that there is more than one dsDNA trapped between the bead and the capillary surface, and therefore such cases are not included in our studies. Successive overstretching cycles lead to RecA nucleation on dsDNA which allows complete coverage of the dsDNA molecule by polymerized RecA within one hour . Under oFor the pulling experiment results shown in Figure MD simulations offer a third tool for considering the stability of dsDNA binding in site I. Though Figure Figure Results of experiments and simulations indicated that the binding in the RecA-heteroduplex complex is more stable and mismatch insensitive than in B-form dsDNA, but they have not provided any reason for that enhanced stability. We sought possible origins by making potential maps of the RecA-heteroduplex complex like those shown in Figure The results presented so far indicate that for some sequences, binding of ssDNA to the presynaptic filament can be much less sensitive to the presence of mismatches than strand exchange. Thus, homology recognition must not simply depend on the mismatch sensitivity of site I base pairing. In order to better understand what does determine homology recognition, we have performed detailed studies of the mismatch-dependent instability of 20 bp interactions containing 17/20 matched bases (Supplementary Table S2). Figure In the strand exchange experiments shown in Figure Importantly, Figure x axis label, k, refers to the sequences with mismatched triplets and corresponds to the separation between the central base in the mismatched triplet and the nearest dsDNA end. The length of the longest region of contiguous homology is m = 19-k. Thus, a triplet at the end has k = 2 and contains 17 contiguous homologous bases. Figure The results shown in Figure x axis has no meaning; however, we wanted to compare the final values for those sequences with those for the mismatched triplets. In order to compare them, Figure For sequences that contained 17/20 matches where the mismatches were not grouped into triplets, the m = 5 is much larger than the values for a central triplet mismatch, tr m, (m = 9), as well as the values for sequences 2+1 and 1+2 . Thus, the asymptotic value of the fluorescence is not simply determined by m, the longest region of contiguous homology or by the number of matched bases within the entire 20-nt sequence.As indicated by the bar graph in Figure In order to provide another check that in the strand exchange experiments the fluorophores are reporting binding along the 20-nt filament rather than a local interaction occurring near the fluorophore tagged ends, we performed an additional set of experiments for each sequence shown in Figure Figure Though annealing showed poor rejection of the mismatched triplet at the 5′ end, the asymptotic fluorescence values seem to suggest that it shows good rejection of a triplet at the 3′ end. We believe that the apparent good rejection of the mismatched triplet at the 3′ end is an artifact due to the partial opening of the B-form dsDNA near the mismatched end where the fluorophore is located. The opening reduces the FRET signal emitted by partially paired dsDNA; therefore, it represents local melting, not global melting. Since in this case the fluorescence signal does not represent the DNA annealing, the bar representing that result is shown with a diagonal pattern to indicate that the fluorescence may not accurately represent the binding.The green bars in Figure in vivo. In vivo, the most probable interaction would involve 5/20 matched bases randomly distributed along the full 20-nt length. Thus, in order to consider the effectiveness of homology discrimination in vivo, we performed strand exchange experiments like those shown in Figure In the previous section we dominantly considered interactions that produce detectable strand exchange products. Though studies of sequences with 17/20 matched bases provide useful information about homology recognition, interactions between sequences with 17/20 matched bases rarely occur Figure M contiguous bp. In the model, the initial test rejects sequences that contain j or more mismatches per M base pair group. For j = 1, only perfectly matched contiguous M base pair groups pass the initial test, whereas for j = 2, the system passes contiguous M base pair groups that contain either 0 or 1 mismatch. A 20-nt sequence contains (21 − M) distinct contiguous M base pair groups.Previous work had suggested that homology recognition might involve an extremely stringent initial test of ∼8 contiguous base pairs, followed by less stringent testing . Such anNj <jMbp with < . For two sequences of equal length aligned in registration, NjM bp with < is the number of distinct groups of M contiguous base pairs that contain less than j mismatches. If there is no group of M contiguous bp that includes less than j mismatches, then NjM bp with < = 0. The model predicts that such an interaction between two 20-nt sequences should not produce detectable strand exchange products, even if the total number of matches per 20 bp is large. In this very simplistic model, the rejection is assumed to be insensitive to the positions of the mismatches in the group. It is also assumed to be insensitive to the nature of the mismatch and the overall composition of the 20-nt sequence. Of course a more realistic model would capture all of these features that we ignored; however, this very simplistic model gives surprisingly good results, as we discuss in detail below.To evaluate the results of the initial tests, we calculated NM bp with <j for M values from 4 to 20 and j values from 1 to 3, as shown in Supplementary Figures S10 and S11. We note that the experimental results are in poor agreement with any initial test that does not accept any mismatches because the rejection would be too stringent. Each separate curve in Figure x axis values correspond to the position of first base in each contiguous 8 group within a particular 20 nt sequence. Thus, the x axis values range from 1 to 13 since within a 20 nt sequence there are 13 possible positions for the first base in a group of 8 contiguous bp. In Figure y axis values correspond to the number of mismatches within the contiguous 8 bp whose first base is located at the position corresponding to the x axis value. Except for the mis4 sequence with 16/20 matched bases (purple curve), none of the sequences include a contiguous 8 bp group with fewer than two mismatches; therefore, for all of the sequences except mis4, N8 bp with <2 = 0. In contrast, N8 bp with <2 = 4 for sequence mis4 since within mis4 there are four groups of 8 contiguous bp that contain less than two mismatches. As Figure To provide quantitative information, we compared the measured final fluorescence values with x axis in Figure x axis in Figure x position corresponds to a particular sequence. In Figure N8 bp with <2 for the same sequences. Comparison of Figure The Comparison of Figure NM bp with <j, we calculated the correlation between the values of N8 bp with <2 shown in Figure M values between 4 and 14 and all j values between 1 and 3. The results of those calculations are shown in Supplementary Figure S12. As discussed above, j = 1 values give very poor fits for any M. For j = 2, the best fit occurs for M values between 6 and 10. The results also show that, for M < 12, an initial test with j = 3 would pass too many interactions; however, j = 3 shows correlations that maximize for M values between 12 and 16. We believe that the peak corresponds to the period doubling of the j = 2 M = 8 peak since the j = 3 M = 12–16 accepts two mismatches in 16 bp (2/14 = 1/7) which is similar to accepting one mismatch in 8 bp.In order to evaluate the relationship between the final value of the fluorescence and Previous work had proposed that since the complementary strand backbone has little favorable interaction with the presynaptic filament, the binding of the complementary strand to the presynaptic filament must be governed by the Watson–Crick pairing of the complementary strand bases with the initiating strand bases . If thisin vivo rapidly form stable homologous products that may incorporate some mismatches?In what follows we will consider two major questions: (i) What are possible origins of the stable mismatch insensitive binding of the complementary strand in the postsynaptic filament? (ii) How can RecA mediated homologous base pairing This work shows that, in the RecA-heteroduplex complex, base pairing is not stressed by interstrand backbone repulsion. Thus, one might expect base pairing in the RecA-heteroduplex complex to be more stable and mismatch insensitive than in B-form dsDNA. We propose that the drastic reduction in backbone repulsion is not the only factor that makes the binding of the complementary strand stable. It is notable that if naked dsDNA is forced to extend to 1.5x the B-form dsDNA length, and the structure has a three nucleotide periodicity, then the minimum energy structure consists of nearly B-form triplets separated by large rises ,31. DiviIn sum, our studies of the RecA-heteroduplex complex structure suggest that the increased stability and mismatch insensitivity result from at least three effects: (i) A decrease in interstrand repulsion. (ii) An electrostatic barrier that pushes the complementary strand backbone toward the initiating strand . (iii) An increase in the stability of the intrastrand stacking due to the intercalation of protein residues.As discussed above, mismatch rejection during strand exchange does not simply depend on the number of matched bases in the 20-nt sequence, or on the maximum number of contiguous base pairs; however, the correlation data suggest that the rejection of mismatched base pairings is consistent with an initial test of ∼8 contiguous bp that accepts up to one mismatch within those 8 base pairs. Though the data require that some single mismatches must pass the initial test, the data do not preclude the possibility that the initial test efficiently rejects some single mismatches.We propose that the rapid initial testing does not take place in structures with dsDNA triplets in the post-synaptic conformation, but rather in transition states like that shown in Supplementary Figure S13, which may not be characterized by the stable mismatch insensitive binding in the heteroduplex. Since the sparsity of accidental matches allows more than 96% of all base pairings to be rejected by an initial 8-bp test that accepts one mismatch, rapid homology testing is consistent with the observed properties of the heteroduplex. Finally, we speculate that the large increase in stability that is observed when strand exchange products extend to 8 bp is the result of a transition to a structure where 8 contiguous bp occupy the position observed in the heteroduplex crystal structure.Previous experimental ,35 and tFigure in vivo, strand exchange does not become irreversible until ATP hydrolysis unbinds the heteroduplex from the filament after the strand exchange product has extended to approximately 80 bp (in vivo, additional factors may promote rejection of non-homologous sequences that are accepted by RecA mediated recognition. Moreover, recombination between divergent sequences can be effectively inhibited by the DNA mismatch repair machinery involving mismatch-directed dissociation of recombination intermediates (Importantly, ly 80 bp . Previouly 80 bp  has showly 80 bp  and the mediates –43.In vivo, a homologous sequence that created a 20-bp strand exchange product would continue to extend the product at a rate of ∼6 bp/s until ATP hydrolysis drives irreversible unbinding of the dsDNA from the filament after the strand exchange product extends to ∼80 bp (For repair and recombination to be rapid and accurate, it is not only important that non-homologous base pairings be rejected, it is also vital that homologous base pairings efficiently form stable strand exchange products. o ∼80 bp .In sum, if the two step kinetic recognition model that we propose is correct the vast majority of mismatched interactions can be very rapidly rejected by an initial test that considers up to 8 bp that can accept up to one mismatch; however, a combination of the probability distribution governing the number of accidental matches present in interactions between two regions of a bacterial genome and the poor stringency shown in the heteroduplex may allow strand exchange to pass through short mismatched regions within homologous interactions while non-homologous interactions are stringently rejected even if they contain large regions of accidental homology. This combination of stringent rejection of non-homologous interactions and acceptance of short mismatched regions within homologous interactions is highly desirable in living systems. The results and analysis in this work are consistent with this recognition model, as is previous work in RecA ,28,29,37Supplementary Data are available at NAR Online.