Nocturnin, a circadian deadenylase, is expressed rhythmically in a wide variety of tissues, but we report here that Nocturnin expression is arrhythmic in epididymal white adipose tissue (eWAT) of mice housed in 12∶12 LD with ad libitum access to food. However, Nocturnin expression becomes rhythmic in eWAT of mice placed on restricted feeding. We show here that Nocturnin's rhythmic expression pattern is not dependent upon feeding, nor is it acutely induced by feeding in the liver or eWAT of ad libitum fed mice. However, Nocturnin is acutely induced by the absence of the expected meal in eWAT of restricted fed mice. A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis. Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes.The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues.  Clock or Bmal1 result in obesity, lipodystrophy and/or glucose homeostasis abnormalities Circadian clocks orchestrate a variety of physiological and molecular rhythms that allow organisms to anticipate and respond to the daily changes that occur in the environment such as light, temperature, and feeding/fasting cycles. Restricted feeding studies have revealed that food can act as a zeitgeber , and have demonstrated that peripheral oscillators can be uncoupled from the master pacemaker located in the suprachiasmatic nucleus (SCN) Nocturnin (Ccrn4l) results in metabolic abnormalities Nocturnin expression peaks in the early night in a variety of tissues such as the liver, the kidney, the spleen, and the retina Nocturnin deficient mice show no abnormalities in circadian behavior nor in the rhythmic expression of core clock genes in the liver, showing that Nocturnin is not part of the core circadian mechanism, but rather contributes to the rhythmic output of the clock Nocturnin−/− mice are resistant to diet-induced obesity and hepatic steatosis, and show dampened rhythms or decreased expression levels of genes related to lipid metabolism in the liver, such as Pparγ and Scd1, respectively The connection between circadian biology and metabolism is not limited to the core clock machinery, but extends to genes downstream of the clock as well. For example, loss of the rhythmically expressed deadenylase Nocturnin is the only known mammalian deadenylase that responds acutely to extracellular stimuli: It is induced in NIH-3T3 fibroblasts by serum and by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and it is also acutely induced by insulin in 3T3-L1 preadipocytes in vivo significance of this acute responsiveness has not been clear.It has also been demonstrated that Nocturnin mRNA levels remain rhythmic with a peak expression time occurring in the early night Nocturnin's rhythmicity in the liver can be driven by systemic cues. However, it is unclear whether Nocturnin's expression pattern in the liver, and perhaps in other tissues, is due to an acute induction by extracellular stimuli, or whether it is the result of an entrained mechanism independent of the oscillation of the local liver clock.In mice that conditionally over-express REV-ERBα in the liver, thus rendering the circadian clock nonfunctional in this tissue, Nocturnin's role in metabolism, its inducibility by extracellular stimuli in vitro, and its capability to be driven by systemic cues in the liver, we set out to examine Nocturnin's expression pattern in white adipose tissue and to investigate whether Nocturnin can be acutely induced by feeding or fasting cues in the liver and the white adipose tissue, two tissues central to metabolic homeostasis. We report that Nocturnin's expression pattern is not rhythmic in white adipose tissue in ad libitum feeding conditions, but is rhythmic in white adipose tissue of mice subjected to temporal, not caloric, restriction of food availability. Also, we show that Nocturnin is acutely induced by fasting in the white adipose tissue of restricted-fed mice.White adipose tissue plays a pivotal role in energy homeostasis by storing energy in the form of triglycerides after a meal, and undergoing lipolysis during times of fasting. It also functions as an endocrine organ secreting a variety of adipokines that have systemic affects on appetite and glucose homeostasis Nocturnin was acutely induced by stimuli such as TPA and insulin in vitro raised the possibility that Nocturnin may be acutely induced in response to feeding Nocturnin expression peaks in the early night in the livers of ad libitum fed mice housed in 12∶12 light:dark (LD) conditions  and forskolin in NIH3T3 fibroblasts  which results in the phosphorylation of hormone sensitive lipase (HSL), perilipin A, and cAMP response element binding protein (CREB), and ultimately the breakdown of triglyceride-storing lipid droplets rst exon , and Nocroblasts . Overexptivation . We nextpression  showing Nocturnin−/− mice have no change in circadian locomotor activity rhythms or in the expression patterns of canonical clock genes in the liver suggesting that Nocturnin is an output of the circadian clock Nocturnin expression is not acutely induced by food nor by the absence of the expected meal in the livers of restricted fed mice suggests that the regulation of Nocturnin expression in the liver is likely the result of the local circadian clock, a food entrained oscillator, a systemic cue that is not dependent upon the daily presentation of food, or some combination thereof. Since Nocturnin shows robust rhythmicity in most tissues where it has been examined Nocturnin is not strongly rhythmic in white adipose tissue under an ad libitum feeding paradigm was surprising. This finding suggests Nocturnin may be uncoupled from the regulation of the core clock machinery or influenced by other signals that are dominant to the clock in this situation.Cry1−/−;Cry2−/− mice Nocturnin becomes rhythmically expressed in the white adipose tissue of restricted fed mice, indicating that its rhythmic expression pattern is driven by the temporal availability of food. Nocturnin's peak expression time in the white adipose tissue of restricted fed mice is six hours later than the peak expression time in the liver, implying that different mechanisms modulate this effect. It has been shown that peripheral tissues respond to food entrainment differently, as the phase of peak Per1 expression between the lung and the liver differ, and the liver entrains to the feeding schedule faster than the lung. It was suggested that this difference could be due to the liver's important role in metabolism Nocturnin expression patterns in the liver and in the white adipose tissue of restricted fed mice suggest that even tissues directly involved in metabolic homeostasis have differing responses to food entrainment. Our data not only demonstrate a tissue-specific expression pattern of Nocturnin, but also demonstrate that the expression patterns of some core clock genes are tissue-specific.A recent study demonstrated that the temporal pattern of feeding, as well as the availability of food, greatly affected the phase of the rhythmic expression of the transcriptome in the liver, and even is capable of partially restoring rhythmic gene expression patterns in the otherwise arrhythmic Per1 and Per2 expression patterns become highly variable in eWAT and are expressed at mid-range levels when compared to the expression patterns in the white adipose tissue of ad libitum fed animals, unlike the liver. In contrast to our results, one previous study has reported rhythmic expression patterns of Per1 and Per2 in eWAT of restricted-fed animals, but perhaps the difference results from the manner in which the studies were conducted Zvonic et al. was less severe (12 hour food availability as compared to our 6 hour feeding window), fewer time points were used within a 24 hour period than were used in our study, and Zvonic et al. placed animals on restricted feeding for seven days instead of twelve to fourteen Bmal1 peak expression occurs three hours earlier in eWAT than in the liver on restricted feeding. Bmal1 expression levels are approximately three-fold higher in the eWAT of restricted-fed animals than in the eWAT of ad libitum fed animals, whereas restricted feeding results in a decrease of peak expression of Bmal1 in the liver. The tissue specific expression patterns of core clock genes in restricted-fed animals raises the possibility that the regulation of their expression may be achieved through unknown mechanisms or may be the result of different entraining signals.In a restricted feeding paradigm, we have shown that Nocturnin is induced by fasting in restricted fed mice is the first example of Nocturnin's acute induction in the mouse. In Drosophila melanogaster it has been shown that curled, the homolog of Nocturnin, is induced by starvation further supporting a role for Nocturnin in fasting Nocturnin's induction is tissue specific and feeding regimen specific, as it only occurs in the white adipose tissue, and not in the liver, under a restricted feeding paradigm. Several possibilities could explain the feeding regimen specific induction of Nocturnin in eWAT. Gene expression in the rat salivary gland entrains to a feeding schedule after a sympathectomy has been performed, indicating that sympathetic input to the tissue modulates food entrainment and perhaps inhibits food entrainment in this tissue Nocturnin's inducibility in white adipose tissue may be suppressed when the phase of the tissue is entrained to the light-dark cycle, and this suppression could be relieved when the tissue phase-shifts in response to a feeding schedule.The observation that Nocturnin in response to fasting may be found in the observation that there are different methods of energy utilization during the different stages of food deprivation. The early fasting stage is categorically centered on glycogenolysis, and after glycogen stores in the liver are depleted, fuel sources are produced by gluconeogenesis and by lipolysis of white adipose tissue Nocturnin seen in restricted fed mice.Another potential explanation for the restricted-feeding specific induction of Nocturnin is the fact that lipolysis in response to an acute stress, such as that presented to the restricted fed mice, is robust, transitory, and predominantly mediated by catecholamines Nocturnin is induced by a rise in cAMP levels, a central step in the well-studied hormone-induced lipolysis cascade. We show that Nocturnin expression increases in response to forskolin, to IBMX, and to the overexpression of PKA in NIH3T3 fibroblasts  Nocturnin plays in white adipose tissue, and how that differs from the role Nocturnin plays in the liver, will add to our knowledge regarding the crosstalk between circadian clocks and metabolism.Mammalian Nocturnin has been shown to exhibit deadenylase activity Nocturnin should also investigate different white adipose tissue depots, as it has been shown that qualitative differences exist between different depots. For example, human visceral white adipose tissue exhibits a stronger lipolytic response to catecholamines than subcutaneous adipose tissue, and the anti-lipolytic affect of insulin is stronger in subcutaneous adipose tissue than visceral adipose tissue Nocturnin, as well as the regulatory mechanisms behind the patterns.Future studies of Nocturnin in the white adipose tissue's response to fasting in restricted fed mice, and highlight the usefulness of Nocturnin and white adipose tissue as tools with which to investigate the intersection of circadian timing and metabolism.It is interesting to entertain the possibility that white adipose tissue could play a prominent role in food entrainment. Given its ability to dynamically respond to food or the lack thereof, its ability to integrate a variety of systemic signals both neural and humoral, and its function as an endocrine organ, it is well positioned to aid the adaptation of the animal to the timing of food availability. Our studies suggest a role for ad libitum feeding to establish whether Nocturnin is rhythmically expressed in white adipose tissue, and to compare that expression profile to the liver as a control. All studies thereafter were conducted in 12∶12 LD to keep the photic stimuli consistent with the experiment conducted in 2 followed by cervical dislocation. For time points conducted during the dark hours, mice were euthanized in the dark, and tissues were collected in the light. Specifically, for studies testing the dependence of Nocturnin's expression on food, mice were housed in 12∶12 LD, with running wheels, and were fed standard chow ad libitum for 7 days. Then food was removed at ZT12 (onset of darkness), and mice were euthanized 25 hours later, at ZT13. Tissues were harvested, frozen in liquid nitrogen, and stored at -80°C until further use. For studies testing the induction of Nocturnin expression in response to feeding after a fast, mice were habituated for 7 days to 12∶12 LD, with running wheels, and with ad libitum access to standard chow. On the eighth day, food was removed at ZT12. On day nine, the cohort was split and one group remained without food while the other group received a meal at ZT2. All mice were euthanized at ZT4, tissues were harvested and frozen in liquid nitrogen, and stored at −80°C until further use. For the restricted feeding experiments, mice were habituated to 12∶12 LD, with running wheels, and with ad libitum access to standard chow for 7 days. At ZT9 on the seventh day, food was removed. Each day, from this day on, food was placed into food bowls in the bottom of the cages at ZT3 and was removed at ZT9. Ad libitum fed control mice had free access to food, but also received the food bowls at the same time and in the same manner as the restricted fed mice. Mice were on restricted feeding for 12-14 days before harvesting tissues every three hours. Tissues were frozen in liquid nitrogen and were stored at -80°C until further use. To test the affect the absence of the expected meal would have on Nocturnin expression, mice were habituated for 7 days in cages with running wheels placed in 12∶12 LD with ad libitum access to standard chow. They were placed on restricted feeding, as described above, for 12 days. On the 13th day, no food was given at ZT3. Tissues were harvested at ZT4 and at ZT7, frozen on liquid nitrogen, and stored at -80°C until further use.All experiments were conducted under the guidelines set forth by the Institutional Animal Care and Use Committee at the University of Virginia. For all experiments, C57BL/6 male mice aged 4–6 months were used. Mice were housed in 12∶12 LD with Tissues were homogenized in Trizol  using Lysing Matrix D  and the FastPrep homogenizer . RNA isolation was conducted according to the Trizol manufacturer's instructions. Total RNA was DNAse-treated , extracted by acidic phenol chloroform extraction  and precipitated with ammonium acetate  and ethanol. 500 ng of total RNA was used to synthesize cDNA using the iScript cDNA Synthesis kit , according to manufacturer's instructions.ad libitum fed mice, and the fasted restricted fed mice were normalized to acidic ribosomal phosphoprotein PO (36B4). The data are graphed and reported as the mean fold change above 36B4 levels (±SEM). CircWave v1.4 software  was used to analyze the rhythmicity of gene expression patterns. p-values reported are the result of an f-test, user defined alpha  = 0.5. Graphs marked with ‡ represent data sets to which no curve could be fit, and all other graphs have a p-value ≤0.05. Forskolin induction data were normalized to Cyclophilin B. Points represent mean and error bars represent the standard error of the mean (±SEM). T-tests assuming equal variances were performed  and the two-way p-values were reported.Real-time PCR was conducted with the Single Color MyiQ Detection System and iQ SYBR Green Supermix . See 2. For induction experiments in NIH3T3 fibroblasts, cells were seeded and grown to full confluence before adding IBMX  or forskolin . The vehicle used to dissolve both forskolin and IBMX was ethanol. Cells were harvested 0, 2, 4, or 6 hours post-treatment by washing the cells once with cold phosphate buffered saline, scraping and lysing with Trizol , and storing the lysates at −80°C until further use. Transient transfections were conducted using FuGene6  and plasmids pCMV-PKA or dominant negative pCMV-KCREB . Cells were collected approximately 24 hours after transfection in the same manner as described before. 3T3-L1 pre-adipocytes were cultured in high glucose-L-glutamine DMEM  supplemented with 10% Fetal Bovine Serum-Premium Select  at 37°C with 5% CO2. Pre-adipocytes were differentiated by growing to confluence and adding Differentiation Media: DMEM-hg-L-glutamine, 10% FBS, 10 µg/mL insulin , 1 µM Dexamethasone , 0.5 mM 3-Isobutyl-1-methylxanthine , and 1 µM Rosiglitazone , set as Day 0. Forty-eight hours later, media was replaced with DMEM-hg-L-glutamine, 10%FBS, 10 µg/mL insulin, 1 µM Rosiglitazone. Every 48 hours media was changed with standard culture media (DMEM-hg-L-glutamine 10%FBS) and 10 µg/mL insulin. Differentiation was confirmed by visual observation of cell morphology and forskolin induction experiments were conducted on Day 10 post-differentiation. For induction studies, 3T3-L1 adipocytes were first serum starved by adding serum free DMEM-hg-L-glutamine for two hours. Then 10 µM forskolin  or vehicle (ethanol) was added to the cells and cells were collected 0, 2, and 6 hours post-treatment. Samples were collected as above and frozen at −80°C until further use.NIH3T3 fibroblasts were cultured in high glucose-L-glutamine DMEM  with 10% Fetal Bovine Serum-Premium Select  at 37°C with 5% CONocturnin sequences were aligned, and the 10 kb region upstream of the translation start site was identified. These sequences were analyzed with MatInspector. Putative sites reported here have 100% conservation of the core sequence and 75% conservation of the matrix sequence.Mouse (AC118601) and human (AC093602) Table S1Primer sequences for quantitative RT-PCR.(DOC)Click here for additional data file.


ASF1A and IL7R, involved in maintenance of chromatin structure and the immune system, associated with familial longevity already in middle-age. The size of this association increased when controls were compared to a subfraction of the offspring that had the highest probability to age healthily and become long-lived according to beneficial metabolic parameters. In conclusion, an “aging-signature” formed of 21 genes was identified, of which reduced expression of ASF1A and IL7R marked familial longevity already in middle-age. This indicates that expression changes of genes involved in metabolism, epigenetic control and immune function occur as a function of age, and some of these, like ASF1A and IL7R, represent early features of familial longevity and healthy ageing.The Leiden Longevity Study consists of families that express extended survival across generations, decreased morbidity in middle-age, and beneficial metabolic profiles. To identify which pathways drive this complex phenotype of familial longevity and healthy aging, we performed a genome-wide gene expression study within this cohort to screen for mRNAs whose expression changes with age and associates with longevity. We first compared gene expression profiles from whole blood samples between 50 nonagenarians and 50 middle-aged controls, resulting in identification of 2,953 probes that associated with age. Next, we determined which of these probes associated with longevity by comparing the offspring of the nonagenarians (50 subjects) and the middle-aged controls. The expression of 360 probes was found to change differentially with age in members of the long-lived families. In a RT-qPCR replication experiment utilizing 312 controls, 332 offspring and 79 nonagenarians, we confirmed a nonagenarian specific expression profile for 21 genes out of 25 tested. Since only some of the offspring will have inherited the beneficial longevity profile from their long-lived parents, the contrast between offspring and controls is expected to be weak. Despite this dilution of the longevity effects, reduced expression levels of two genes,  Nonagenarians and centenarians delay or escape age-related diseases Previous studies have investigated whether there are gene expression changes that occur with age in brain, lymphocyte, kidney and skeletal muscle tissues Here we report transcriptional profiling of whole blood samples from participants of the Leiden Longevity Study which is based in the Netherlands and comprises nonagenarian sibling pairs, their middle-aged offspring and the partners of the offspring as population controls. A comparison of gene expression profiles between the nonagenarian siblings and controls identified profiles that associate with age. The subsequent comparison of the middle-aged offspring and the controls enabled the identification of genes that mark the propensity to become long-lived in middle-age.Gene expression profiles were generated from 150 whole blood total RNA samples collected from 50 families belonging to the Leiden Longevity Study (LLS). From each family, one nonagenarian sibling, one of their offspring and the offspring's partner  were proThe explorative analysis is divided in two parts . The fir−10) is located in the leucine rich repeat neuronal 3 gene  and showed a 3.1-fold decreased expression in nonagenarians, which was also the largest difference in expression level between the two groups. The largest increase in expression level was 2-fold for a probe targeting the interferon, alpha-inducible protein 27 isoform  gene locus. This probe could also target SYTL1, but since the levels of a specific probe for SYTL1 did not correlate with the IFI27 probe (Pearson correlation = −0.14), while a unique probe for IFI27 did correlate (Pearson correlation = 0.96), the result suggests that IFI27 mRNA is responsible for the association. Average expression levels of the 2,953 probes show a distribution over the whole range of measured intensities  consortium The nonagenarian participants in the LLS each exhibit the longevity phenotype and their offspring, as a group, carry the potential to become long-lived as demonstrated by beneficial physiological characteristics and decreased morbidity of that generation ZNF331, 19q13.33) whereas the largest decrease in expression level was for a probe targeting a mRNA at chromosome 1q43 (no known corresponding gene); this had a 2.1-fold decreased expression in offspring every ten years relative to controls. The only probe that demonstrated a significant increase in expression with age in the offspring (a 1.3-fold change every ten years) targeted an EST at 20q13.2 . We observed no differential expression between offspring and controls for LRRN3 and IFI27, which had the most extreme changes with age in the first analysis in which nonagenarians and controls were compared.The most significant differentially expressed probe  corresponded to the zinc finger protein 331 gene  and the gene different between offspring and controls (i.e. ZNF331). We added 22 genes that were differentially expressed in the offspring compared to controls which additionally associated with the concept “cell aging” in literature using the text-mining tool Anni 2.0 http://biosemantics.org/anni/. In total 25 genes  gene, were selected for replication analyses.To replicate our results we measured the expression levels of a subset of genes via RT-qPCR in 79 nonagenarians, 332 offspring and 312 controls . We firs25 genes , includiLRRN3 again showed the largest significant decreased expression in the nonagenarian siblings and IFI27 the largest increased expression. The analysis to test for replication of the novel samples only (58 nonagenarians and 281controls) resulted in the same observations  have previously been reported to associate with age in a microarray study of T cell mRNA IFI27 and LRRN3 genes. IFI27 encodes interferon alpha-inducible protein 27 whose biological function is currently unknown. Leucine rich repeat neuronal 3 (LRRN3) is mainly involved in activation of MAPK activity and endocytosis MYC, WRN and MTOR. The MYC protein is a transcription factor that regulates transcription of specific target genes and overexpression of the MYC gene has been associated with a variety of cancers WRN gene cause the Werner progeria syndrome, an autosomal recessive disorder characterized by premature aging and genetic variation in or near this gene have been associated with several age-related diseases and survival MTOR gene encodes a serine/threonine kinase and its downregulation is associated with extended lifespan in model organisms The genome-wide expression analysis between nonagenarians and controls resulted in the identification of 2,953 probes associated with age. RT-qPCR replication experiments with a large sample size resulted in replication of 21 out of 25 genes identified by the microarray analysis as differentially expressed with age. Five of these genes (The main cellular pathways that changed with age in the microarray dataset were ‘response to stimulus’ (including ‘immune response’ and ‘response to stress’), ‘signal transduction’, ‘gene expression’ and ‘protein binding’. These GO terms have previously been found to associate with aging, suggesting that these are systemic age-related processes. The only pathway that associated with longevity at middle-age in the offspring-control comparison was the ‘Rho protein signal transduction’ pathway, which is part of the GO term ‘signal transduction’. The Rho family of GTPases are small GTPases that regulate a wide variety of processes in the cell including growth, cytoskeletal organization, transcription and lipid metabolism Research into human familial longevity and healthy ageing is complex in the sense that there are no controls from the same birth cohort to compare to long-lived persons since such controls would have died twenty years ago. We investigated offspring as a proxy for the nonagenarian case group since these can be compared to controls from the same birth cohort. However, in the offspring the longevity phenotype will undoubtedly be diluted as compared to the nonagenarians, since only a part of the offspring will become long-lived and a part will age comparable to controls. The consequence of the dilution of the longevity cases is that effect sizes are underestimated. Indeed the effect size of the association with longevity increased when the healthiest offspring was compared to the controls. Future follow-up data on age of death will reveal which offspring carries the longevity phenotype.In this study we investigated expression profiles in whole blood samples of participants because blood is an easily accessible tissue. This allows us to investigate the large sample sizes required to detect small effect sizes. An advantage of using blood compared to other tissues is that cell subsets can easily be measured and used to select samples or used as covariates in analyses whereas different cell subsets present in other tissues are difficult to quantify and can not be taken into account in any analyses. In our microarray study we selected samples from offspring and controls with similar cell counts. Furthermore, since aging affects the whole organism and since blood is in contact with all tissues, blood may reflect in part the physical health of the whole body. Disease state is mirrored by gene expression profiles in blood ASF1A and IL7R marked familial longevity already in middle-age and the effect size was enhanced in the subset of longevity family members with a beneficial metabolic marker profile. Functional and longitudinal studies are necessary to establish which of these genes are true biomarkers for healthy ageing and which contribute causally to this trait. Our findings illustrate that gene expression changes occurring as a function of age may partly represent early detectable features of human longevity and healthy ageing.In conclusion, we identified a transcriptional signature in whole blood consisting of 21 genes that repeatedly differentiated between nonagenarians and middle-aged controls. The expression level of two of these genes, The individuals investigated in this study are participants of the Leiden Longevity Study. The families participating in this study have at least two siblings with a minimum age for men of 89 years and for women of 91 years One long-lived sibling, one of their offspring and the partner of the offspring were selected from 50 families for the current study . These tThe 150 samples that met the RNA quality criteria were hybridized onto 54k CodeLink Human Whole Genome Bioarrays . cDNA synthesis, amplification, biotin labeling and hybridization onto the microarrays were performed according to the manufacturer's instructions using the Codelink iExpress reagent kit (cat. No. 67601000). The slides were scanned with a MicroArray Scanner G2505B scanner  and the image was quantified with the CodeLink Expression software (version 4.2).http://www.bioconductor.org). After normalization, we used log2-transformed expression intensities for all subsequent analyses. Raw and normalized microarray data are stored in the GEO online database record GSE16717 in compliance to MIAME guidelines. A principal component analysis (PCA) was performed on all samples  and hybridization date was identified as a confounding factor causing a deviation in the data, which was attributed to a scanner maintenance check during the measurements of the samples (data not shown). Therefore, all subsequent analyses were adjusted for hybridization date coded as the 14 days of hybridization as categorical variable, which was sufficient to adjust for this technically induced variation. Since samples were randomly hybridized, no confounding with group was present. None of the other tested parameters, like RNA quality, isolation date and time of blood draw appeared to be a significant confounder of the expression data.Raw intensities were background subtracted, set to 0.5 when results were negative and normalized using the Cyclic Loess method in the Codelink software package The 54,243 probes on the CodeLink Bioarray were newly annotated to Entrez Gene ID's and GO identifiers in two steps. First, all probe sequences were mapped to Unigene and dbEst sequences with BLAT while allowing for, at most, one mismatching nucleotide. Subsequently, all Probe to Unigene annotations were transformed to probe to Entrez Gene ID and probe to GO ID annotations using Entrez Gene-to-Unigene and Entrez Gene-to-GO ID mappings available on the ftp server of NCBI. Probe-to-EST annotations were treated in a similar way, except that ESTs were first mapped to RefSeq Gene IDs by aligning ESTs to RefSeq exons using galaxy and the genomic alignments of ESTs and RefSeq genes from UCSC (hg18). All EST to RefSeq mappings with a sequence similarity >95% were maintained for further mappings of ESTs-to-Entrez Gene IDs using the Entrez Gene-to-RefSeq Gene ID mappings available at NCBI. All information used was downloaded in October 2008, using versions NCBI Build 36.1 or UCSC hg18.Probes without a “Good” flag indicating that the mRNA is detected, as determined by the CodeLink Expression software, in at least 10% of the samples  and/or probes without at least a known chromosome band location according to the new annotation  were excluded from the analysis, resulting in 45,164 remaining probes.ijY is the base 2 logarithm of the expression level of probe j in sample i, iGroup is the group (0 for control or 1 for long-lived nonagenarian) of subject contributing sample i, iGender corresponds to the gender of the ith sample , iHyb is the categorical term of hybridization day on which the sample i was measured and εij represents an error term. The coefficients βj1 represents by how much gene expression increases between the groups, βj2 represents the change in expression for a female in comparison with a male sample, βj3 represents the change of expression across hybridization dates, and 0jβ represents the baseline regression level of the probe, for male control samples on the first hybridization date. Resulting p-values were adjusted for multiple testing using Benjamini and Hochberg's False Discovery Rate (FDR) method All single gene analyses were performed using the Limma (Linear models for microarray data) package in R iGroup corresponds to the offspring/control status of the ith sample  and βj1 is the change of expression with group status. For each probe j, we determined the coefficient with respect to group status (βj1).To find longevity-related differences between offspring and controls, the following model was used:i*AgeiGroup corresponds to the slope of expression with age of the jth probe set for offspring or controls, i*GenderiAge corresponds to the slope of expression with age of the jth probe set for males or females, i*GenderiGroup indicates the interaction of group and gender of the jth probe set for the ith sample, βj4 represents the change of expression with age for offspring in comparison to control, βj5 represents the change of expression with age for a female in comparison with a male sample, βj6 represents the change of expression with gender between offspring and controls. The interaction between group and offspring in comparison to control  was determined and resulting p-values were corrected for multiple testing using the FDR method.To test differences in biological aging rate in offspring and controls, we used the following model:The Globaltest methodology was designed to determine whether the common expression pattern of genes within a pre-defined set is significantly related to clinical outcome When performing pathway analysis on Model 3 we used GlobalAncova, which is a method similar to Globaltest suited for models including interaction terms −ΔΔCt method, in which Ct indicates cycle threshold, the fractional cycle number where the fluorescent signal reaches detection threshold To confirm the accuracy of the measured expression profiles, we compared the expression level of 25 probes from the CodeLink Bioarrays with corresponding Taqman® assay  All serum measurements were performed with fully automated equipment. For glucose, triglycerides, total cholesterol, HDL-cholesterol, adiponectin and free triiodothyronine, the Hitachi Modular or the Cobas Integra 800, both from Roche, Almere, the Netherlands were applied. CVs of these measurements were all below 5%. Lipoprotein particle sizes have been analyzed in 165 families from the Leiden Longevity Study using a 400-MHz proton NMR analyzer at LipoScience.ASF1A and IL7R was compared to the levels in 312 controls using linear regression. In this analysis, expression level was the dependent variable and the two groups of individuals (offspring vs. controls) were included in the model as a categorical variable together with age and gender and their interaction as covariates. To take into account dependencies within sibships, robust standard errors were used, i.e. the variance was computed from the between family variation. P-values were also based on these robust standard errors. Analyses were performed using the software package STATA/SE 11.0 .To select that subfraction of offspring with the highest probability to age healthily and become long-lived because of their metabolic risk profile in middle-age, we identified the subjects, separately for men and women, within the lower 5% tail of the distribution for glucose, triglycerides, free triiodothyronine, ratio of total cholesterol over HDL cholesterol and the FRS. Additionally we identified for LDL particle size and adiponectin those subjects, separately for men and women, within the upper 5% tail of the distribution. This resulted in a total of 78 offspring out of 332 from which gene expression levels of Figure S1Distribution of expression intensities of 2,953 age-related probes. The x-axis indicates average log2 expression intensities of all samples; the y-axis indicates the log2 fold change between nonagenarians and controls. Black circles represent all 45,164 probes; red filled dots represent the 2,953 age-related probes with a FDR≤0.05.(TIF)Click here for additional data file.Table S1Significant gene expression changes with chronological age and/or familial longevity. A total of 2,953 probes whose expression level changed significantly between controls and long-lived nonagenarians (regression modeling with FDR multiple testing correction) are shown. The table also indicates the chromosomal location of the probe. “FC” indicates the fold change between groups, a FC above 1 indicates increased expression in nonagenarians compared to controls, and a FC below 1 indicates decreased expression in nonagenarians compared to controls. MULTIPLE indicates a probe annotated to more than three chromosomal locations.(XLS)Click here for additional data file.Table S2GO terms found significantly differentially expressed between nonagenarians and controls. Globaltest pathway analysis resulted in 109 Gene Ontology terms of which the expression of the involved probes differed between long-lived nonagenarians and controls (FWER≤0.05). Gene Ontology categories include biological process (BP), molecular function (MF) and cellular component (CC).(XLS)Click here for additional data file.Table S3List of probes of which the gene expression changes with age significantly differed between offspring and controls. A total of 360 probes whose expression level changed significantly with age between offspring and controls (regression modeling with FDR multiple testing correction) are shown. “FC” indicates the fold change between offspring and controls per 10 years; a FC above 1 indicates a larger slope of expression as a function of age in offspring, a FC below 1 indicates a larger slope of expression as a function of age in the controls. MULTIPLE indicates a probe annotated to more than 3 chromosomal locations.(XLS)Click here for additional data file.Table S4RT-qPCR results of replication samples only. RT-qPCR results of replication samples only are shown. “FC” indicates the fold change between groups; a FC above 1 indicates an increase in expression and a FC below 1 indicates a decrease in expression compared to the controls. “p” indicates the unadjusted p values. Bold indicate p values are below the significant level of 0.05 after Bonferroni correction for multiple testing.(DOC)Click here for additional data file.


The progress of PD and its related disorders cannot be prevented with the medications available. In this study, we recruited 8 PD and 4 PD plus patients between 5 to 15 years after diagnosis. All patients received BM-MSCs bilaterally into the SVZ and were followed up for 12 months. PD patients after therapy reported a mean improvement of 17.92% during “on” and 31.21% during “off” period on the UPDRS scoring system. None of the patients increased their medication during the follow-up period. Subjectively, the patients reported clarity in speech, reduction in tremors, rigidity, and freezing attacks. The results correlated with the duration of the disease. Those patients transplanted in the early stages of the disease (less than 5 years) showed more improvement and no further disease progression than the later stages (11–15 years). However, the PD plus patients did not show any change in their clinical status after stem cell transplantation. This study demonstrates the safety of adult allogenic human BM-MSCs transplanted into the SVZ of the brain and its efficacy in early-stage PD patients. Paralysis Agitans) or Parkinson's disease (PD) was originally described by James Parkinson in 1817 as “Involuntary tremulous motion, with lessened muscular power, in parts not in action and even when supported; with a propensity to bend the trunk forwards, and to pass from a walking to a running pace: the senses and intellects being uninjured”  for CD34 and CD45.μL cell suspension were incubated in the dark for 30 min at 4°C with saturating concentrations of phycoerythrin PE-conjugated antibodies. Appropriate isotype-matched controls were used to set the instrument parameters. After incubation, cells were washed three times with wash buffer and resuspended in 0.5 mL wash buffer for analysis. Flow cytometry was performed on a 5HT Guava instrument. Cells were identified by light scatter for 10000 gated events and analyzed. The following markers were analyzed: CD34-PE, CD45-PE, CD73-PE, CD105-PE, CD166-PE, and CD90-PE .Immunophenotyping of the cultured BM MSC was performed using flow cytometry to identify the presence of specific cell-surface antigens. Briefly, BM MSCs were dissociated with 0.25% trypsin-EDTA and resuspended in wash buffer at a concentration of 1 × 10 cells/mL. Cell viability was measured by flow cytometry using 7-amino actinomycin D (7-AAD) 200 The trilineage differentiation capacity of human BM MSC into osteoblasts, adipo cytes and chondrocytes was investigated to confirm mesenchymal properties. Briefly osteoblast differentiation was induced by culturing human BM MSC in Stempro Osteogenesis Differentiation kit  for 15 days as per the recommendations provided by the manufacturers. Fresh medium was replenished every 3 days. Calcium accumulation was assessed by Von Kossa staining. The differentiated cells were washed with DP BS and fixed with 10% formalin for 30 min. The fixed cells were incubated with 5% silver nitrate for 60 min under ultraviolet (UV) light and then treated with 2.5% sodium thiosulphate for 5 min. Images were captured using an Nikon Eclipse 80i microscope .μL Oil Red O staining solution was added and incubated for 10 min at room temperature. The cells were rinsed five times with distilled water. The images were captured using a Nikon Eclipse 80i microscope . For chondrogenic differentiation, human BM MSC were cultured for 21 days using chondrogenesis differentiation kit  as per the manufacturer's recommendations and stained with Safranin O as specified. The images were captured using Nikon Eclipse 80i microscope .To induce adipogenic differentiation, human BM MSC were cultured for 21 days using Adipogenesis differentiation kit  as per the protocol recommended by the manufacturers. Medium was replenished every 3 days. Cells were fixed in 10% formalin for 20 min, and 200 in vitro propagation of BM MSC, these cells were karyotyped prior to transplantation. The chromosomes were visualized using a standard G-banding procedure, and more than 200 cells were analyzed per sample and reported according to the International System for Human Cytogenetic Nomenclature (ISCN).To rule out any chromosomal aberrations during Prior to dispatching the cells for transplantation, a battery of in-process quality testing was performed on the cells. These include morphology, immunophenotyping cell surface marker analysis, endotoxin testing using LAL test, and mycoplasma using RT-PCR was also done. Only those cells fulfilling the ISCT criteria for MSCs were released for transplantation.Any sample positive for endotoxin and mycoplasma was discarded immediately and appropriately.The final cell suspension which was provided to the clinician for transplantation was again tested for cell surface marker analysis as mentioned above. In addition, karyotyping, endotoxin, and mycoplasma were also performed as mandatory quality testing. Cell viability was measured by flow cytometry using 7AAD (7-amino actinomycin D). Certificate of analysis (COA) was prepared, and cells were released along with documentation for transplantation.Subjects, both male and female between 18–80 years, were enrolled for this study. The patients were screened for HIV, HBV, HCV, CMV, and VDRL followed by inclusion criteria selection before participating in the trial. 8 PD patients and 4 PD plus syndrome patients were chosen for the trial  have been classified under PD plus syndrome patients). This would help us understand the role of bone-marrow-derived mesenchymal stem cells in the early stages of the disease and in rapidly progressing PD plus syndrome patients.Those patients who fulfilled the following inclusion and exclusion criteria were included for the study.Should be in the age group of 18–80 years.Should be fully conscious, alert, and oriented while providing consent.Should show significant motor and nonmotor symptoms.Subject should provide a written informed consent and agree to return for follow up.Subject should be clinically diagnosed for Parkinson's disease and PD included disorders with motor complications despite adequate oral anti-Parkinsonian therapy.Should be able to comply with and understand the required visit schedule.Patient is suffering from Dementia (MMSE < 25).The extent or severity of the disease is not measurable.If the subject suffers form preexisting medical conditions such as bleeding disorders, and septicemia.Patients with a past (within one year) or present history of psychiatric disorder.If the subject has been enrolled in other investigational drug trial or has completed any trial within the last 3 months.If hemoglobin < 10 gm/dL, serum creatinine < 2 mg/dL, serum total bilirubin < 2 mg/dL, and HbA1c < 7%.Pregnant or nursing or women in child bearing age without adequate contraception.The subject tested positive for HIV, HCV, HBV, CMV, or VDRL.These patients were admitted 48 hours prior to the procedure, and a detailed clinical evaluation was performed including UPDRS, MMSE, gait and neuropsychological assessments. General physical examination and cardiac status of the patients were evaluated before inclusion into the study. MRI was done at the baseline and at 12 month follow up. MR Tractography was conducted at 3rd, 6th and 9th month follow up sessions.b value: 1000 frequency: 128, phase 128, NEX-1, slice thickness: 5 mm with zero interslice gap and bandwidth: 250 kHZ).MRI imaging was done on a 1.5 5T  machine. Routine imaging was done with axial FLAIR and T2W images and Sagittal T1W images. DTI of brain was done in the axial plane using an 8-channel CTL array spine coil with the following parameters-25 directions EPI tensor imaging .t-test  has been used to find the significance of study parameters on continuous scale within each group.Descriptive statistical analysis has been carried out in the present study. Results on continuous measurements are presented on Mean ± SD (min-max) and results on categorical measurements are presented in number (%). Significance is assessed at 5% level of significance. Student As mentioned in the earlier study  the cellThe patient was positioned supine for the transplantation and the parts aseptically prepared. Under short propofol anesthesia bilateral frontal burr holes were drilled and small dural openings made. The sub ventricular zone was accessed through a standard brain cannula with CRW stereotactic frame or Stealth (Medtronic) navigation assistance. BM-MSCs, at a dose of 2 million cells/kg body weight, were transplanted into the brain and gelfoam placed over the dural defect prior to closing of the wound.After operation the patients were observed in the neurointensive care unit for 24 hours following which they were shifted to the ward and discharged home on the 4th/5th day.The patients were followed up closely every three months for one year. They were assessed by an independent neurophysician and a movement disorders specialist. During each visit the patient was clinically examined, UPDRS score performed, neurologically assessed, and medications reviewed. At the final follow-up visit, that is, 12th month the patient would undergo a MRI scan to check for any structural changes in comparison to the baseline scan. The medication would be reviewed at each visit and adjusted based on the symptoms. Any adverse event would be reported to the concerned investigator and IEC.In this study, 12 patients were recruited according to the study design as per the inclusion and exclusion criteria mentioned above. This included 9 males and 3 females in the age group of 37–69 years. The duration of the disease varied between 3 and 15 years in the study group. Out of the 12 patients, 4 were diagnosed as PD plus and belonged to the older age group. The details of the patients who participated in this study are mentioned in 2 million cells/kg body weight suspended in 2 mL of saline was implanted bilaterally into the subventricular zone using burr hole surgery technique. All patients tolerated the procedure well, there were no postoperative complications and were discharged within a week's time from the hospital. This indicates that there were no immediate cytotoxic effects due to implantation of allogenic bone marrow mesenchymal stem cells in to the sub-ventricular zone of the brain and the procedure was safe.3 adult healthy screened donors were recruited for the aspiration of bone marrow under general anesthesia. BM-MSCs were isolated and cultured as described in . These c−/CD45−/CD73+/CD90+/CD105+/CD166+ as depicted in The cells displayed a typical spindle shaped fibroblast-like appearance as shown in In order to ensure that the BM-mesenchymal stem cells maintain their typical properties, the trilineage differentiation capacity of these cells was demonstrated. The cells were found to undergo adipogenic, osteogenic and chondrogenic differentiation as determined by Oil Red O stain, Von Kossa stain and Safranin O stains, respectively,  will be available online at doi: 10.1155/2012/931902).This set of analysis confirms that the cells being used for the clinical study are truly mesenchymal in nature.ex vivo propagation. A representative ideogram is shown in the Supplementary data .All the samples used for transplantation were processed for karyotyping prior to transplantation by a trained cytogeneticist. No abnormalities/aberrations were noted after Clinical assessment was performed on all patients based on 4 basic parameters of the UPDRS scoring system: (1) mental behavior and mood, (2) activities of daily living, (3) motor disabilities and impairment, and (4) complications of PD therapy. This was considered as the primary measurable outcome of the clinical study. The scoring was typically done during the “off” period (approx.12 hours off the anti-Parkinsonian medication) and during the “on” period (within 1-2 hrs of the medication) where maximum benefit could be appreciated in the PD symptoms. The average score during the “on period” at baseline was 62.33 and after stem cell transplantation it improved to 51.16 that is, an improvement of 17.92% over the baseline . SimilarMost of the PD patients reported subjective improvement during the first follow up that is, at 3 months after stem cell transplantation. These include clarity in speech, reduced tremors, and rigidity, and general sense of well being. These changes were seen in the later follow ups too indicating that the changes were not transient but more permanent. Similar improvements were also noted for some of PD plus patients but not all. However, for these patients the changes were transient and by the next follow up (6th month) most of them had progressed further into the disease.As depicted in It needs to be mentioned that the PD plus patients could not be rated using the UPDRS scoring system after stem cell transplantation due to the severity and progression of the disease. Hence, this data has not been mentioned.MRI of the brain was done before and 12 months after stem cell therapy as shown in The results of MR tractography have been shown in Tables The dosage of anti-Parkinsonism medication before and after stem cell transplantation was analyzed. For 4 PD plus patients, the clinician recommended an increase in dosage of medication based on their progression of the disease. However, for the PD patients in the early stages of the disease similar increase in dosage was not required. The dosage has remained the same as the baseline medications prescribed. This indicates that the disease has not progressed further after stem cell transplantation. Only for 2 patients, the dosage of Syndopa had to be increased. This is probably because the disease had already advanced beyond repair at the time of stem cell transplantation which is also evident from the UPDRS scores of the patients.Therefore, out of the 8 PD patients, intervention was in the early stages in 6 patients. The progression of the disease appears to have been slowed after the administration of stem cells. They did not require enhancement of dose. In the late stages of disease and PD plus patients, stem cell transplantation had shown relatively lesser symptomatic relief and on the other hand needed an increase in medications.Care givers have noticed an overall improvement in their activity levels in 7 patients, which includes reduction in tremors both at rest and in motion, better clarity in speech, reduction in rigidity, ability to walk for longer distances and perform personal tasks independently. This has a significant impact on the well being of the patient and further substantiates the fact that the progression of the disease has been slowed down after stem cell therapy.in vitro can be transdifferentiated into functional dopamine secreting cells [The current treatment for PD includes pharmacotherapies and deep brain stimulation techniques. Lesioning surgery is gradually fading. However, these can only produce symptomatic relief and have their own limitations and long-term side effects. Therefore, the need for alternative therapy is the need of the hour. Fetal nigral striatal grafts and neural stem cells are successful candidates used in the last few decades as a choice for PD. However, due to the fetal source it has ethical, immunological, tumorigenic risks besides sourcing concerns . This hang cells , 21, 22.ng cells –25. HencIn our first study , we havein vitro much earlier (Passage 3) and hence it was challenging to be able to upscale the cells for transplantation (unpublished data). The mixed results obtained may be attributed to the differences in the cell properties observed.During the earlier study we also noted that there is a difference in the population doubling time (PDT), morphology, differential potential, and cell senescence. Although the cells met the required standards of the ISCT, the PDT, and cell surface marker expression and differential potential was observed to be lesser than healthy donor BMMSCs. This may be attributed to the higher age of the patient where cells are known to have shorter telomere length  and lowein vitro, maintain differential potential and cell surface marker expression. It is believed that these cells will have potentially higher therapeutic benefits. Since these cells can be produced in a large scale, cell expansion would also help to make the therapy more affordable. And bilateral transplantation would prevent any further degeneration on the contralateral side.In view of the preceding results, in this study we wanted to understand the safety and feasibility of bilateral “allogenic” bone-marrow-derived mesenchymal stem cells for PD. The rationale was to rule out bone marrow aspiration in the aging population of PD patients and the morbidity associated with it. In the current study, we have transplanted allogenic healthy donor mesenchymal cells at passage 2. These cells are easy to upscale After receiving appropriate approvals, the study was conducted in 8 PD patients and 4 PD plus patients. The small number was chosen to understand the safety of injecting allogenic adult bone marrow mesenchymal stem cells into the subventricular zone of PD patients.Parkinson's disease involves both the nigral and extranigral systems. As a result, there are motor complications, associated dementias, multiple system dysfunctioning, and decline in cognitive functions with time.Most studies have focused on the motor aspects only which are due to the loss of dopaminergic neurons (DA) in the substantia nigra of the midbrain. Cell replacement experiments conducted till date are targeted towards the replacement of the DA neurons. The results of fetal mesencephalic transplantation show graft induced dyskinesias due to inflammation around the implants, mixed population of DA neurons, and inappropriate synaptic contacts , 8–10.Therefore, in our study we opted to choose a cell type which would primarily help in neuroprotection of the affected region irrespective of the cell type, followed by neurogenesis. This is a more global approach to the problem which would target not only the classical motor symptoms but also associated memory loss and decline of cognitive functions. Our primary aim was to forestall the progress of the disease and secondly help in restoration of neural functions. This is the first paper to demonstrate that bilateral allogenic transplantation of adult bone-marrow-derived mesenchymal stem cells is safe and has beneficial neuroprotective and neurorestorative effects in PD patients.In this context, it is imperative to understand how mesenchymal stem cells help in neuroprotection and neurogenesis.It is well established that BMMSCs are capable of releasing cytotrophic mediators such as nerve growth factor (NGF) superfamily-Brain-derived neurotrophic factor (BDNF) and NGF-3; Glial-derived neurotrophic factor (GDNF) and neurturin . These nα, interleukin 1β and γ interferon in the SN of PD patients [In vitro studies show that they are involved in immunosuppressive activities although the mechanism of action needs further clarity. In autoimmune encephalomyelitis animal models, mesenchymal stem cells have demonstrated a reduction in inflammatory infiltrates, lesser relapses, and neural insults. Recent studies are suggesting that NSAIDs are said to have beneficial effects in PD patients. An anti-inflammatory beneficial effect of mesenchymal stem cells is being proposed here.Data from animal studies also demonstrate that inflammation at the SN of the midbrain leads to significant loss of dopaminergic neurons. Also there is a noted increase in the levels of tumour necrosis factors-patients . MesenchThe neural stem cells are located in the subventricular zone of the brain and hence is the most preferred site of injection in this study although invasive. The exogenous BMMSCs would help to activate and increase proliferation of the resident stem cells which has regenerative capacities (endogenous regeneration). At the same time, SVZ is far away from the known lesioning targets in the brain.In this study, we report that there is 22% improvement in the UPDRS scores of the patients treated. The improvement was noticed only in the early diseased patients and not in PD plus. At the end of the study (12 months) it was not required to increase the medication which is an indicator that disease progression has been prevented. Further follow-up studies are on-going. No study till date has reported the ability to stall progression of PD in patients. Concurrent to the UPDRS scores, there was a sense of subjective well being perceived by the patient and caregivers in 10 out of 12 patients.de novo and hence none of the drugs or surgery are useful. Unfortunately cell therapy does not seem to alter the course. This creates a need for further studies where we need to consider the option of providing multiple doses of cells at frequent intervals and/or test the potential of stem cells derived from a different source like adipose tissue or umbilical cord matrix as these are also known to possess neuroprotective effects and higher transdifferentiation potentials. At baseline and 12 months after stem cell transplantation, MRI of the brain was performed. Although there has been improvement in symptoms and no further progression of the disease, there were no structural changes observed in the MRI scan. The MR tractography results show a specific pattern of recovery. Certain structural changes were observed in the genu of the corpus callosum and the left peduncle suggesting that early regeneration of the tracts probably occurs here. These changes are persistent throughout the follow-up study and clinically correlated with improvements reported in the patients. Currently we are continuing the study, with PET scans which will give us valuable information on any metabolic and functional changes happening in the SN region of the midbrain. This would give us further valuable clues in to the mechanism of regeneration in the human PD-affected brain.For PD plus, 3 patients have shown slight transient improvement post stem cell transplantation. In 1 patient there was no noticeable change. We feel that BM-mesenchymal stem cells should be considered as a treatment of choice in early-stage PD patients to appreciate maximum benefits. This is due to the loss of the “neuroregenerative reserve” present in the brain. It proves the fact that once the disease has progressed further and involves multiple areas, it is difficult to stop the process. Also the degeneration is so extensive and rapid that it is beyond the reparative capacity of the exogenous bone marrow mesenchymal stem cells to help in neuroprotection and neurogenesis. We also assume that the degeneration process is slow in the initial phases and gains momentum with time. PD plus appears to be involving multiple areas of degeneration in situ neuroregenerative reservoir of stem cells is present that is, in the early part of the disease.Thus, to summarize, BM-mesenchymal stem cells have a three pronged therapeutic approaches in neurodegenerative diseases such as PD: neurogenesis, neuroprotection and neural plasticity. Also, it is essential to expose the degenerating brain to the exogenous stimulus of stem cells, while the This is the first paper to demonstrate that bilateral allogenic transplantation of adult bone-marrow-derived mesenchymal stem cells is safe and has beneficial neuroprotective and neurorestorative effects.The study establishes the safety of adult bone-marrow-derived mesenchymal stem cell transplantation bilaterally into the subventricular zone of the human brain using burr hole surgery. There are improvements in the UPDRS scores of the PD patients, reported subjective well being and no increase in medications during the follow-up period. It is to be noted that no improvements were observed in the PD plus patients. This strengthens the fact that stem cell transplantation in the early stages of PD has the potential to prevent further progress of the disease. Results from this study suggest that allogenic BM-mesenchymal stem cells may be used as a disease modifying therapeutic strategy in treating PD. Unlike the known indications for surgical intervention, we recommend intervention in the early part of the disease to reap the best benefits. However, further long-term follow-up studies need to be carried out to understand the long term safety and sustainability of the benefit. Currently studies are going on to elucidate the mechanism of action of these cells in neuroprotection and neurogenesis in PD-affected human brain.


With approximately 2.5 billion people at risk, dengue is a major international public health concern. Dengue vaccines currently in development should help reduce the burden associated with this disease but the most efficient way of using future dengue vaccines remains to be defined. Mathematical models of transmission can provide insight into the expected impact of different vaccination strategies at a population level and contribute to this definition.We developed and analyzed an age-structured, host-vector and serotype-specific compartmental model, including seasonality. We first used this transmission model to identify the immunological interactions between serotypes that affect the risks and consequences of secondary infections  and reproduce the observed epidemiology of dengue. For populating this model, we used routine surveillance data from Southern Vietnam and the results of a prospective cohort study conducted in the same area. The model provided a good fit to the observed data for age, severity of cases, serotype distribution, and dynamics over time, using two scenarios of immunological interaction : short term cross-protection alone (6–17 months) or a combination of short term cross-protection with cross-enhancement . Finally, we explored the potential impact of vaccination for these two scenarios. Both highlighted that vaccination can substantially decrease dengue burden by reducing the magnitude and frequency of outbreaks.Our model suggests that seasonality and short term cross-protection are key factors for explaining dengue dynamics in Southern Vietnam. Vaccination was predicted to significantly reduce the disease burden, even in the situation where immunological cross-enhancement affects the risks and consequences of secondary infections. Flavivirus genus, circulate in tropical and subtropical regions of the world and are transmitted to humans by mosquitoes, primarily Aedes aegypti and Aedes albopictusDengue disease is caused by four distinct serotypes of dengue virus. The viruses, members of the The transmission dynamics of dengue has generated a lot of scientific interest, partly because primary exposure to one serotype is thought to alter the course of secondary, hetero-serotypic infections. The first mathematical model of dengue transmission, designed by Fischer and Halstead There are other differences between published models of dengue transmission. With the notable exception of the agent-based model developed by Focks et al. With the aim of understanding the potential impact of a dengue vaccine on a population, we explored the characteristics of a mathematical model of the transmission of the four dengue serotypes and its ability to reproduce the epidemiology observed in Southern Vietnam.A mathematical model representing the transmission dynamics of the four dengue serotypes in the human and mosquito populations of Southern Vietnam was developed and analyzed. Here we focus on the main features of the model and the epidemiological data used for its validation. Building upon a host-vector model of dengue that focused on the interactions between two serotypes We assumed that all infants are born susceptible to the four dengue serotypes, and that maternal antibodies play no role. While maternal antibodies affect the risk of contracting dengue during infancy, the impact of this simplifying assumption on the overall disease dynamics is likely to be minimal Each infection is serotype-specific and provides lifelong, serotype-specific immunity, i.e., each individual can develop up to 4 dengue infections during their lifetime. The level of infectiousness of human hosts to biting vectors is likely to vary between severe, mild and asymptomatic infections as it depends upon viral load.Consistent with the work reported by Bartley et al. We designed the model to be flexible to consider different types of immunological interactions between serotypes: temporary or permanent cross-protection, temporary or permanent cross-enhancement, or a combination of cross-protection and cross-enhancement.Cross-protection was assumed to start immediately after the infectious period and prevents individuals from becoming infected by another serotype for a period ranging from 6 months to lifelong. In some scenarios, cross-protection was assumed to prevent only symptomatic disease but not asymptomatic infection.Cross-enhancement was assumed to start immediately after the cross-protection period, or immediately after the infectious period when no cross-protection is considered. Three forms of cross-enhancement can be considered: increased susceptibility to infection by another serotype; increased disease severity of secondary infections , and increased infectiousness (through differences in infectiousness according to disease severity). We tested several scenarios for each form, corresponding to different levels of cross-enhancement in order to identify those likely to provide a good fit to observed data . However, since we excluded the possibility of co-infection by several serotypes, the vast majority of compartments therefore remained empty. For instance for all scenarios considering temporary cross-protection only 176 states in each age group contribute to dengue dynamics in the absence of vaccination and 1691 when vaccination is implemented. This simplified model excludes highly unlikely situations, such as the simultaneous co-infection by the 4 dengue serotypes.Human demography was modeled according the methods adopted by Hethcote Like Bartley et al. To parameterize and then validate the model, data describing different epidemiological variables from a single country or area are required. We chose to focus on Southern Vietnam, as for this area a comprehensive series of case reports is available, together with the results of a recent cohort study of disease and infection in children.Dengue is endemic throughout Vietnam, but transmission is highest in the south where very large epidemics occur regularly The available surveillance data  include:The rate of dengue infection, which is central to our transmission model, cannot be derived from surveillance data. For this purpose, we used the results of a prospective cohort study conducted in Long Xuyen, the capital of capital of An Giang province in the Mekong Delta  [34]. ThTo ensure consistency between the two datasets, cases detected in the cohort study were classified as DF, DHF or DSS according to the case definitions used in the Vietnamese national surveillance program, which are derived from World Health Organization (WHO) guidelines published in 1997 Finally, in addition to dengue surveillance data and the results of this prospective cohort study, we used demographic information and data on the duration of infection in humans and in mosquitoes to populate our model  was 90% or 70%.We focus here on the main insights provided by the model after exploring various scenarios and comparing model outputs with observations. The first calibration step enabled us to rule out as unrealistic several scenarios for which the model outputs were incompatible with the observed average incidence, including permanent cross-protection and high degrees of cross-enhancement . We therefore limited our exploration of interaction scenarios to a plausible range . Short term cross-protection against infection  was found to be critical to reproducing the observed periodicity  versus 41%–45% for models only including seroprotection.We also considered a scenario where the first two infections conferred complete protection against any further dengue infection. This scenario generated longer cycles (18 years) and smaller annual variations 11%  than obsIn summary the calibration procedure led us to select six scenarios: 3 that only include cross-protection between serotypes (Models S2.2A–S2.2C) and 3 that combined cross-protection and cross-enhancement (Models S2.4A–S2.4C). These six models were used to explore the impact of vaccination programs.The impact of demographic changes was explored (models 2.6A–B) by considering a sudden change in the population growth rate for both the human and mosquito populations (similar sized change for each population) with the model S2.4A used as a reference. Doubling the population growth rate of both host and vector led to a higher frequency of outbreaks (median duration of outbreak cycles 3 years), a decrease in the average age of dengue cases (14.3 years to 10.5 years) and an increase in dengue incidence (+80%). When population growth was set to zero, which corresponds to an ageing population, the median cycle duration increased from 6 to 12 years, dengue incidence decreased by 50% and the mean age of cases increased to 21.5 years.A sudden reduction in the size of the vector population, such as that caused by the implementation of a vector control program, reduces the disease incidence in the short term, but does not alter the long-term dynamics of transmission. For example, a 50% reduction for 2 years (model S2.6E) led to a dramatic, 96% reduction in dengue incidence measured over 5 years. However, the reduction over 10 years was only 20%, and became null over a 15 years period. The reduction in incidence seen in the short term is cancelled out in the long term by a higher incidence after the program’s interruption.Disassociating the growth rate of the human and vector populations had a much stronger impact in the long run. Stopping the vector population growth, while allowing the human population to continue growing (model S2.6C), eliminated the circulation of dengue after 20 years. Doubling the vector population growth rate on the other hand resulted in more frequent outbreaks and a tripling of the dengue incidence (median duration of cycles 3 years [2.4–3.8]).Routine vaccination of 90% of 1 year-olds  with 90%Due to indirect effects, vaccination benefits both vaccinated and non vaccinated individuals: the reduction of the lifetime risk of infection was respectively 87% and 96% for vaccinated and non vaccinated individuals with models S2.2B, respectively 77% and 90% with model S2.4B.Decreasing the assumed vaccine efficacy to 70% results in similar changes to the transmission dynamics  but provLowering the vaccination coverage rate from 90% to 70% also results in a smaller reduction in disease incidence: According to the scenario considered, the reduction ranged from 78% to 82% when the vaccine efficacy was 90% and the duration of protection set at 30 years.Decreasing the assumed average duration of protection from 30 to 10 years had little effect over the first 10 years  but reduModels including cross-enhancement (S2.4A–C) globally led to a smaller reduction in dengue incidence than models that only considered cross-protection (S2.2A–C). For a vaccine efficacy of 90%, duration of protection of 10 years, and 90% coverage, the overall reduction in incidence was 64–70% for models S2.2A–C and 54–55% for models S2.4A–C. Models including cross-enhancement also led to a slightly lower reduction of severe cases than mild cases (respectively 51–52% and 58–62%).Differences between models S2.2A–C and S2.4A–C were more pronounced when efficacy and coverage were set at 70% and duration of protection at 10 years. In this case, dengue incidence was reduced by 38%–44% and 24%–25%, respectively. Conversely, very limited differences were observed when the average duration of full protection is set at 30 years and efficacy and coverage at 90%: respectively 90–93% for models S2.2A–C and 87–90% for models S2.4A–C.Finally, we also analyzed the impact of demographic changes on vaccination benefits. If the absolute benefit of vaccination is directly affected by a demographic change that impacts dengue incidence, the relative benefit remains relatively stable. For instance, if the average duration of full protection is set at 30 years and efficacy and coverage at 70%, dengue incidence is reduced by 57% if the population grow at a high rate (model S2.6A), by 54% if the population stop growing (model S2.6B) or if the population continue growing at the rate currently observed (model S2.4C).We built a model capturing most of the features likely to influence the dynamics of dengue transmission and, after validating it against epidemiological data from Southern Vietnam, tested different hypotheses for their ability to reproduce the observed epidemiology of dengue in this highly endemic area. Our results highlight the critical role of short-term cross-protection that desynchronizes the epidemic cycles of the four dengue serotypes, resulting in 5–6 year-cycles consistent with those observed in Southern Vietnam. Wearing and Rohani reached the same conclusion after comparing a model with Thai data Our model suggests that the duration of cross-protection is in the range 6–17 months but did not point to a specific duration. However, our model assumption that this duration follows an exponential distribution may be inaccurate since this implies large variation on the duration of cross-protection from one individual to another. As dengue is a seasonal disease in southern Vietnam, it is plausible that cross-protection of shorter duration, associated with a narrower temporal distribution would suffice to desynchronize epidemics. Cross-protection against disease symptoms alone was insufficient to reproduce the observed multiannual cycles but this result was obtained for a scenario in which asymptomatic infections significantly contribute to disease transmission.In our model, cross-enhancement without cross-protection did not reproduce the observed epidemiology of dengue in Southern Vietnam. This, however, does not invalidate the existence of immunological cross-enhancement that has been considered in many dengue transmission models These analyses also provide insights into other aspects of the transmission dynamics of dengue. Consistent with findings reported by Wikramaratna et al. By exploring the effect of the relative size of the human and vector populations, we saw that temporarily reducing the size of the vector population had only a short term effect of the transmission dynamics. We equate this with the impact of temporary vector control activities, some of which have had short term success but have not succeeded in maintaining disease reductions in the long run Our model was sensitive to the disassociation of the growth rate of the human and vector populations. This can be equated with lifestyle or environmental changes, such as urbanization or global warming, which have consequences on the ecology of the vector population and thereby on dengue transmission Our analysis was based on two complementary sources of information: routine surveillance in Southern Vietnam and a prospective cohort study with active surveillance conducted in the area There are, however, some limitations to the combined use of these data sources. The data collection period differed between the two sources Click here for additional data file.Text S2Supplementary results.(DOC)Click here for additional data file.


Current treatments for osteochondral injuries often result in suboptimal healing. We hypothesized that the combination of hyperbaric oxygen (HBO) and fibrin would be superior to either method alone in treating full-thickness osteochondral defects.Osteochondral repair was evaluated in 4 treatment groups  at 2-12 weeks after surgical injury. Forty adult male New Zealand white rabbits underwent arthrotomy and osteochondral surgery on both knees. Two osteochondral defects were created in each femoral condyle, one in a weight-bearing area and the other in a non-weight-bearing area. An exogenous fibrin clot was placed in each defect in the right knee. Left knee defects were left empty. Half of the rabbits then underwent hyperbaric oxygen therapy. The defects in the 4 treatment groups were then examined histologically at 2, 4, 6, 8, and 12 weeks after surgery.The HBO+fibrin group showed more rapid and more uniform repair than the control and fibrin only groups, but was not significantly different from the group receiving HBO alone. In the 2 HBO groups, organized repair and good integration with adjacent cartilage were seen at 8 weeks; complete regeneration was observed at 12 weeks.HBO significantly accelerated the repair of osteochondral defects in this rabbit model; however, the addition of fibrin produced no further improvement. Successful repair of full-thickness defects in articular cartilage has been a difficult goal to achieve. Spontaneous repair often fails to completely fill the defect and the new tissue is composed of fibrocartilage rather than the superior hyaline cartilage ,2. AlthoExogenous fibrin clots have been used to facilitate healing in canine and equine knee joints -17. SuchThe study was designed to compare 4 methods of repairing full-thickness cartilage defects: no treatment, fibrin alone, HBO alone, and HBO plus fibrin. All the authors certify that our institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with the principles of ethical research. Table Forty male New Zealand white rabbits with an age of 4 to 5 months old and weighing about 3 kg each were purchased from a licensed dealer. The animals were housed in our animal facility and were fed ad libitum. All animal procedures were performed according to the regulations of the authors' institute.Each animal was anesthetized before surgery with an intramuscular injection of 10 mg/kg ketamine. Both knees were then arthrotomized using a medial parapatellar approach. Two full-thickness defects 3 mm in diameter and 3 mm in depth were drilled through the articular cartilage into bleeding subchondral bone in the trochlear groove of each femur. One hole was made in the center of trochlea that articulated with the patella, a weight-bearing surface; the other was made in the nonarticulated notch area, a non-weight-bearing surface  were randomly chosen to begin hyperbaric oxygen treatment, which was given 5 days a week for 4 weeks . These treatments were given in a hyperbaric animal research chamber . In this compressed air chamber, 100% oxygen was delivered at 2.5 atmospheres absolute (ATA) for a duration of 120 min, using an intermittent schedule of 25 min of oxygen breathing and 5 min of air breathing.The articular defects were evaluated at 2, 4, 6, 8, and 12 weeks after HBO therapy (Table Four groups of specimens were examined. Each group consisted of 20 knees. The left knees (with empty defects) of the non-HBO-treated animals were the control for the other 3 groups. The group with fibrin clots only was composed of the right knees of the non-HBO-treated animals. The left knees of HBO-treated animals were used to evaluate the effects of HBO alone on cartilage repair; the right knees of these animals served as the experimental group treated with both fibrin plus HBO.All specimens were examined by the same 2 observers (LCP and ACC), both of whom were blinded to the treatments used. The grading scale used was a modification of the method of O'Driscoll ,17,20,21P value of ≤ 0.05 was considered to indicate statistical significance.Mean scores for each time period were calculated from the average of the total scores of all 4 specimens in each group, and were expressed as mean ± standard deviation. The Kruskal-Wallis 1-way analysis of variance by ranks test was used to analyze differences between groups. When the Kruskal-Wallis test indicated a significant difference between groups, selective comparisons between the HBO+fibrin group and the other groups were performed using the Mann-Whitney rank sum test. A Gross examination of the surfaces of the defects showed that although the defects in the central, weight-bearing, area of the trochlea were completely covered by 2 weeks after surgery in all animals, it was not until 8 weeks after surgery that the defects in the peripheral, non-weight-bearing, area were completely covered in all animals.The data for each histological category at each time point for all 4 groups are shown in Table At 2 weeks, the HBO plus fibrin group exhibited significantly better filling (category 1) of defects (p = 0.021) than the control group, which had only sparse cellular infiltration in the repaired tissue at that time point. At 4 weeks, the HBO plus fibrin group showed complete filling (category 1) with good integration (category 6). At 6 weeks, significant differences in the histological grading in each category were noted between the HBO plus fibrin group Figure  were almAt 12 weeks, the HBO plus fibrin group showed complete regeneration, proteoglycan staining that was similar to that of normal cartilage (category 4), and a homogeneous distribution of mature chondrocytes, while the control and fibrin groups showed only incomplete fibrous repair, and fibrillation and irregularity of the surface architecture Figure , 8 and 9There were no significant differences in histological grading scores between the 2 HBO groups  at any time point.Because fibrin and hyperbaric oxygenation have both been shown to improve wound healing, but by different mechanisms, we hypothesized that a combination of the 2 would have an additive effect on healing. However, this was not the case. HBO treatment was better than fibrin treatment, which in turn was better than no treatment. Nonetheless, adding fibrin clot treatment to hyperbaric oxygen treatment resulted in no significant additional benefit over hyperbaric oxygen alone.In the current experiment, each treatment was used to treat defects in both the weight-bearing and non-weight-bearing areas of the cartilage surface. As was the case in previous research , the nonAlthough HBO clearly resulted in better repair than fibrin alone or no treatment, we did not test the repaired defects to establish whether the strength was normal. We also did not determine whether the collagen in the cartilage in the repair was type II or the inferior Type I variety. In addition, we did not conduct a longitudinal study to investigate whether the repair would deteriorate with time. Also, it is difficult to achieve integration of repair tissue with normal surrounding tissue. Although our subjective visual impression was that the repairs produced by HBO treatment were well integrated with adjacent cartilage, we have no quantitative data to support this.Since vascular endothelial factors and neovascularization has been observed in the young growing cartilage , the impFull-thickness cartilage repairs proceed in the following sequence: local bleeding and hematoma formation, migration of mesenchymal stem cells from the underlying bone, transformation of these cells into chondrocytes, proliferation of chondrocytes, synthesis of type I collagen, and filling of the defect with fibrocartilage rather than the physicochemically superior hyaline cartilage that is normally present . This pr2, but proteoglycan aggregation is maximal at 3% O2 [2 , but macromolecular synthesis peaks at >13% O2 [2 concentrations of 12-15%, while inhibition of cartilage and type II synthesis occurs at very high (> 90%) and very low (< 5%) oxygen concentrations; and reactive oxygen species (which can be produced by hyperbaric oxygen) stimulate proteoglycan synthesis in chondrocytes at low concentrations and inhibit it at high concentrations [2 tension is needed for wound healing [In addition to increasing neovascularization, hyperbaric oxygenation may directly affect the mixture of growth factors necessary for mesenchymal stem cell migration or the subsequent regenerative events. Unfortunately, the effects of HBO on tissue regeneration are difficult to determine because they do not all occur at the same oxygen concentrations. For example, in in vitro studies, glycosaminoglycan synthesis in bovine growth plate chondrocytes peaks at 21% Oat 3% O2 ; cell pr healing ; howeverFibrin clots in wound care in animal experimental models are believed to serve as a scaffold for repair of an osteochondral defect and to contain chemotactic and mitogenic factors that stimulate cellular elements crucial to tissue healing . With thHBO treatment in this study resulted in a clear improvement in cartilage repair and, unlike other treatments, is completely noninvasive. We do not know if the repaired cartilage is as strong as normal cartilage, or if it will deteriorate over time; nor do we know if HBO therapy will work with defects that do not extend to the bone (the majority of defects), where the mesenchymal stem cells are present. These partial-thickness defects do not regenerate. However, the results reported here do increase hope that a clinically noninvasive method to induce cartilage regeneration will be developed.In conclusion, our results show that hyperbaric oxygen treatment is clearly superior to no treatment in hastening cartilage repair and producing histologically superior cartilage. Packing the defects with fibrin clots was less effective than HBO, and produced no additional improvement when added to hyperbaric oxygenation.The authors declare that they have no competing interests.ACY conceived the idea of the study, performed part of the literature review, and contributed to the drafting of the manuscript. MSL performed part of the literature review and assisted in analyzing the data. SSL assisted in animal surgery and in manuscript drafting. LCP contributed to the interpretation of the light microscopic study. SWU contributed to manuscript editing. All authors have read and approved the final manuscript.


Iron is an essential nutrient for almost every living organism because it is required in a number of biological processes that serve to maintain life. In humans, recycling of senescent erythrocytes provides most of the daily requirement of iron. In addition, we need to absorb another 1–2 mg Fe from the diet each day to compensate for losses due to epithelial sloughing, perspiration, and bleeding. Iron absorption in the intestine is mainly regulated on the enterocyte level by effectors in the diet and systemic regulators accessing the enterocyte through the basal lamina. Recently, a complex meshwork of interactions between several trace metals and regulatory proteins was revealed. This review focuses on advances in our understanding of Cu, Zn, and Ca in the regulation of iron absorption. Ascorbate as an important player is also considered.  In humans, iron is the carrier of oxygen by means of hemoglobin in erythrocytes and myoglobin in myocytes, and is therefore indispensable for providing oxygen to the tissues. Severe iron deficiency leads to anemia with a number of symptoms such as tiredness, palpitations, and other signs associated with impaired oxygen transport. Also, iron overload is linked to several diseases involving functional changes of hemoglobin, such as thalassemia and sickle cell disease. Iron in excess is detrimental because of its propensity to cause oxidative damage. Except for the sloughing of epithelial cells, which removes iron confined in the absorptive cells, there is no other way to eliminate iron. It is therefore of major importance to tightly regulate iron absorption.Iron (Fe) is the second most abundant metal on earth and is a necessity for all life. Iron plays the key role in numerous enzymatic reactions due to its ease in shifting between the two common oxidation states, ferrous  and liver (hemoglobin). Heme iron is coordinated to a porphyrin ring deeply hidden inside a globular protein, which makes the access to iron restricted to small molecules such as Osorption . The dieAn average individual contains about 50 mg Fe/kg, which is approximately 3.5 g for a 70 kg-person [+-coupled folate transporter suggesting that absorption of heme-iron is affected by folate availability [Heme-bound iron may be absorbed from the gastrointestinal lumen through the heme-carrier protein HCP1. HCP1 is primarily a Hlability . The abslability  in orderlability , but the+) and ferrous iron (Fe2+) [2+ or also transports other metal ions [2+ in preference to Zn [2+ is preferred to Fe2+ [2+ is assured by the expression of a brush border-associated ferric reductase. DMT1 (NRAMP2/SLC11A2/DCT1), the main transporter for divalent cations, transports elemental iron across the lumenal membrane of absorptive cells. Human DMT1 (hDMT1) simultaneously imports hydrogen (Hn (Fe2+) . It is stal ions . Althougce to Zn ,9,10,11. to Fe2+ . DMT1A a to Fe2+  suggesti3+ to Fe2+ by means of an electron donated from ascorbate in the cytosolic compartment. It has been observed that DCYTB co-localizes with DMT1 at the lumenal membrane, and the levels are increased in iron-starved cells in the rat [3+ could be transported across the enterocyte membrane by means of an integrin-mobilferrin-paraferritin (IMP) complex in the rat [Duodenal cytochrome b (DCYTB/CYBRD1) has been proposed as responsible for the reduction of ferric iron in humans . DCYTB i the rat  and in h the rat . It has  the rat . In cont the rat . In this the rat . This wo2+ and is then oxidized to Fe3+ by a membrane-bound ferroxidase, hephaestin. The ferric iron is transferred to transferrin (Tf) for systemic transportation.In the cytosol, newly absorbed iron initially joins the labile iron pool (LIP) also referred to as the chelatable iron pool, which consists of metabolically active forms of iron mainly loosely associated with macromolecules. Depending on the cellular requirements, iron may be distributed from the LIP to the major intracellular iron storage protein, ferritin, where iron is stored inside the hollow core as ferric phosphate salts. The ferritin molecule consists of L and H subunits, in which the H subunits possess ferroxidase activity required for the acquisition of iron salts. It has been proposed that a recently identified cytosolic iron chaperone, PCBP1 (hRNP/α-CP1), is responsible for the incorporation of iron to ferritin . HoweverSystemic iron homeostasis must be rigorously controlled, not only because iron is essential for survival, but also since iron is highly toxic if overloaded. Hepcidin, a small peptide hormone produced in the liver, is the key regulator of systemic iron levels. Hepcidin controls iron release from the enterocytes into the systemic circulation by binding to the iron exporter ferroportin, thereby subjecting ferroportin to ubiquitin-dependent degradation . RecentlXenopus laevis oocyte expression system have revealed that ZIP14 imports Fe2+ in addition to Zn, Cd and Mn. Interestingly, the transport of Fe2+ was dependent on Ca2+, but not on Zn2+ [Systemic iron is also absorbed in hepatocytes as nontransferrin-bound iron (NTBI), which requires the usual route of reduction to ferrous iron by membrane-bound reductases and uptake through metal transporters. DMT1 is generally expressed in all tissues and may therefore play a role in hepatocyte iron influx. The zinc transporter ZIP14 is abundantly expressed in liver cells and has been shown to be involved in NTBI uptake . Later s on Zn2+ . The aut on Zn2+ . When ZIIn summary, there are several indications of the involvement of ZIP14 in the regulation of hepcidin control of iron release. There are other zinc transporters currently under investigation, e.g., several zinc importers  in rat liver cells have been shown to be affected by iron status, but their role in regulating iron absorption is unclear .Hepcidin is an important mediator in the acute phase reaction of inflammation . SeveralMacrophages play an important role in executing the regulatory events leading to changes in systemic iron levels. Senescent or damaged erythrocytes are removed from the circulation by phagocytosis. Heme-iron is transported from the phagocytic vesicles into the cytosol by means of a transmembrane permease, HRG1 . ElementThe expression of iron transporters is regulated on the mRNA level by means of common motifs, iron responsive elements (IREs) . Ferriti−/− mice, thus compensating for its absence [There are two forms of IRPs; IRP1 and IRP2. Both IRPs are RNA-binding proteins. IRP1 also function as a cytosolic aconitase and it appears that this is its normal state in animal tissues. The mRNA binding of IRP1 does not increase in iron-deficient mice, despite the activation of IRP2 . In our  absence . The IRPTwo of four isoforms of DMT1  and one It is well known that individuals exposed to a copper-deficient diet develop iron deficiency anemia as well as iron overload in specialized tissues such as the liver and intestine. The iron and copper homeostases are linked by the inability to export Fe in the absence of Cu to the systemic circulation. However, Cu deficiency does not affect ferroportin expression . Ferropo+ across the apical border of the intestinal epithelium. Recent data indicate that DMT1 does not transport Cu+ [+, and has been proposed to require the assistance of the ferrireductase Dcyb, which also functions as a cupric reductase [+ and Fe2+ availability is linked on the lumenal level in addition to the systemic level, further strengthening the co-regulation of these two micronutrients  . The incAscorbate is an important regulator of iron transport; it assists DCYTB in the intestinal lumen by reducing ferric iron to the bioavailable ferrous form. In addition, cellular ascorbate status influences enterocyte protein levels of DMT1 and DCYTB in the absence of iron . HIF2α, Iron down-regulates its own uptake by the DMT1 transporter while zinc up-regulates uptake of iron by increasing the protein levels of DMT1 in human Caco-2 cells . The basThe Zip4 transporter is expressed along the intestine and is crucial for zinc absorption and a normal intestinal epithelium . However2+ [2+ inhibits Fe2+ transport non-competitively with low affinity. It is not clear how Ca2+ interferes with iron transport, but voltage dependence or intracellular Ca2+ signaling seems to be ruled out [2+ after a long exposure time (16–24 h) [2+ incubations (1.5 h) of human intestinal Caco-2 cells down-regulated ferroportin levels at the basal membrane and the associated iron efflux [Calcium is generally believed to interfere with iron absorption. It has also been established that this effect requires high Ca intake and occurs at the lumenal level. The mechanism involves the iron transporter hDMT1, of which transport is inhibited by Ca2+ . Ca2+ inuled out . Iron up16–24 h) . The memn efflux . The ferSeveral studies in humans have shown a correlation with Ca and decreased Fe absorption. However, long-term high Calcium intake does not correlate with impaired iron status, as concluded by Lönnerdal (2010) in an extensive review on the subject .In recent years, substantial progress in the understanding of iron absorption has been made. It is clear that the absorption of iron is extremely complex and it is becoming increasingly difficult to separate the absorption by several players into individual events; when can we actually state that an iron deficiency is primary? We are still lacking pieces of information, which may be the explanation for discrepancies encountered. New studies are frequently published in the field, giving hope that gaps eventually will be filled. However, plenty of additional work is required. + is central to iron absorption and that there is no competition with Fe2+ for DMT1 transport. Also, the reduction of both ions by DCYTB shows that Cu+ and Fe2+ uptake into the enterocyte is co-regulated. In addition, Cu is required for the efflux of Fe2+ through ferroportin, further indicating that their absorption is mutually inclusive. It is clear that CuZinc deficiency has been extensively investigated particularly due to its immune modulatory function and its appearance in other micronutrient deficiencies. Zinc stimulates iron absorption but does not seem to be a prerequisite to maintain normal iron levels. Cow’s milk was, or still is, widely used as a food supplement for small children and is associated with low iron absorption. This may not be an effect of the calcium content. On the mechanistic level, neither zinc nor calcium seem to be as crucial for iron absorption as copper, whilst copper deficiency and sufficient copper levels in the diet have been the least investigated. The same refers also to ascorbate and its intracellular effects on iron absorption.