Ákos Putics

Dalma Vödrös

Marco Malavolta

Eugenio Mocchegiani

Dr Csaba Sőti

Zinc supplementation boosts the stress response in the elderly: 1 Hsp70 status is linked to zinc availability in peripheral lymphocytes 2 3

Keywords: stress response, Hsp70, zinc, zinc supplementation, lymphocytes 21

Chaperones and zinc are indispensable for proper immune function. All the zinc status, 2 the immune function and the stress response decline during aging. Here we studied the effect 3 of nutritional zinc and zinc homeostasis on the stress response in healthy old subjects 4 recruited during the ZincAge European Union project that either underwent or not a 48-day 5 zinc supplementation. Inducible Hsp70 levels were determined at basal conditions as well as 6 after heat shock in the CD3+ and CD3-subset of lymphocytes by a two-color FACS analysis. 7

Short term zinc supplementation resulted in a marked increase in both basal as well as stress-8 induced Hsp70 levels in lymphocytes from healthy elderly donors with a higher impact on 9 CD3+ cells. Heat inducibility showed a strong correlation with basal Hsp70 level, and both 10 basal as well as stress-induced Hsp70 highly correlated with intracellular zinc availability. In 11 conclusion, short-term oral supplementation with zinc safely and efficiently induces the stress 12

response in lymphocytes of old donors. The stress response may be a candidate pathway 13 connecting zinc deficiency with aging and immunosenescence. Thus, proper dietary zinc 14 intake may emerge as a chaperone inducer and an anti-aging mechanism in the immune 15 system. 16 Zinc is one of the most important trace elements in the body [START_REF] Stefanidou | Zinc: a multipurpose trace 5 element[END_REF]. 2

It has a catalytic/regulatory role in many enzymes, maintains the structural integrity of various 3 proteins (such as superoxide dismutase and zinc-finger transcription factors) and modulates 4 protein-protein interactions. At the cellular level zinc is essential for cell proliferation and 5 survival, contributes to genomic stability and antioxidant defense, which highlights its crucial 6 role in aging and age-dependent degenerative diseases. Zinc is indispensable for proper 7 immune function. Zinc deficiency increases susceptibility to infections, compromises both 8 innate immunity and the T-cell compartment [START_REF] Shankar | Zinc and immune function: the biological basis of altered 12 resistance to infection[END_REF]Rink and Haase, 9 2007). It is well documented that zinc status and immune function declines with aging, and 10 zinc supplementation is beneficial to immune responses in the elderly (Haase et al., 2006b;11 Larbi et al., 2006). 12 Molecular chaperones are conserved and abundant proteins that guard the 13 conformational homeostasis of proteins [START_REF] Hartl | Molecular chaperones in cellular protein folding[END_REF]. They maintain signaling, regulate 14 proliferation, differentiation and apoptotic pathways (Sőti et al., 2005b; Sreedhar and 15 [START_REF] Sreedhar | Heat shock proteins in the regulation of apoptosis: new 3 strategies in tumor therapy: a comprehensive review[END_REF]. Chaperones (or stress proteins) confer cytoprotection and assure survival 16 upon various stresses. The stress response is regulated by the heat shock transcription factor 1 17 (HSF-1) 1 , named after the archetype of proteotoxic stress. HSF-1 induces the transcription of 18 various stress proteins. Inducibility of the major chaperone, the 70 kDa heat shock protein 19 (Hsp70) reflects the robustness of the stress response, which is indispensable for adaptation 20 [START_REF] Voellmy | On mechanisms that control heat shock transcription factor activity in 20 metazoan cells[END_REF]. Both a single heat shock as well as transgenic Hsp70 induces longevity 21 [START_REF] Tatar | Chaperoning extended life[END_REF], HSF-1 overexpression induces a twofold life-span extension, while HSF- 1 22 knock-out markedly shortens life-span [START_REF] Hsu | Regulation of aging and age-related disease by 14 DAF-16 and heat-shock factor[END_REF][START_REF] Garigan | Genetic 22 analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and 23 bacterial proliferation. Genetics 161, 1101-1112. zinc in peripheral blood mononuclear cells[END_REF], demonstrating 23 the central importance of a robust stress response in aging in invertebrate models. HSF-1 24 knock-out mice neither display heat shock response, nor show premature aging in 'sterile'

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M A N U S C R I P T 4 laboratory conditions. However, they are hypersensitive to endotoxemia, which reinforces the 1 role of the stress response in immune function and inflammation [START_REF] Xiao | HSF1 is required for extra-embryonic development, postnatal growth and 24 protection during inflammatory responses in mice[END_REF]. 2 Moreover, both chaperone inducibility and chaperone function decrease during aging and in 3 chronic inflammation characteristic to the elderly (Nardai et al., 2002;Sőti and Csermely, 4 2003; [START_REF] Arslan | Protein homeostasis and molecular chaperones in 9 aging[END_REF], therefore preserving their function is an attractive target in anti-5 ageing therapies (Sőti et al., 2005a;Sőti and Csermely, 2005). 6

Zinc and chaperones are connected in many ways [START_REF] Larbi | The heat shock 20 proteins in cellular aging: is zinc the missing link?[END_REF]Arslan et al., 7 2006;Sőti and Csermely, 2005). Zinc is a potent inducer of Hsp70 in cell culture (Hatayama 8 et al., 1993) including lymphoblasts form old, but not from young donors (Ambra et al., 9 2004). Both zinc deficiency and overdose inhibits the stress response in rodent thymus ( Moore 10 et al., 2003). Thus, proper dietary zinc is critical for mounting a robust stress response. The 11 ZincAge project funded by the 6 th Framework Program of the European Union aimed to study 12 the role of nutritional zinc in healthy aging and in immunosenescence. We were interested to 13 study the relationship of zinc homeostasis and aging with the stress response, an essential 14 adaptive and survival mechanism in the immune system. Here we report on the effect of zinc 15 status on the stress response of lymphocytes obtained form healthy elderly donors.

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M A N U S C R I P T 5

Materials and methods 1

Subjects and zinc supplementation 2

Twenty healthy old subjects (between 64 and 85 years of age) were enrolled according 3 to the inclusion criteria required by the ZincAge European specific targeted research project 4 (www.zincage.org) approved by the respective National Ethical Committees. Briefly, all 5 subjects were originated from Italy, lived an independent life, were in a good general health 6 conditions devoid of functional impairment and serious acute or chronic disease, without 7 taking medical drugs or nutritional supplements. All subjects gave informed consent and 8 underwent medical examination and clinical laboratory tests. Seven persons from the twenty 9 healthy old subjects underwent an oral zinc supplementation which was performed with 10 10 mg pure zinc/day (in the form of 50 mg zinc-aspartate Unizink 50, KÖHLER PHARMA 11 Corp., Alsbach-Hähnlein, Germany) for 48 ± 2 days. All subjects admitted to zinc 12 supplementation had values of plasma zinc below 11 M at least at one test out of two made 13 within one year. 14 15

Blood withdrawal and PBMC preparation 16

Heparinized whole blood samples after an overnight fast were withdrawn from each 17 subject before and after zinc supplementation. The whole blood was centrifuged at 450 x g for 18 10 min at 4°C to separate the plasma. Peripheral blood mononuclear cells (PBMC) were 19 prepared by Ficoll-Hypaque (d = 1.077 g/ml) gradient centrifugation (450 x g for 30 min at 20 20°C), washed twice in PBS, and aliquots were made in fetal bovine serum containing 10% 21 DMSO and were frozen in liquid nitrogen until further use. 22

A C C E P T E D M A N U S C R I P T 6
Plasma zinc levels were analyzed by induction plasma coupled mass spectrometry 1 (ICP-MS). All plasma samples and standard were diluted 1:10 with a solution containing the 2 following reagents: 0.1% Triton-X100, to maintain a stable emulsion with the diluted sample 3 and 0.15 % HNO 3 , to ensure that trace elements are maintained in solution and to aid washout 4 of these elements between samples. External calibration solutions containing zinc (blank to 5 2000 ppb), were prepared by serial dilution of parent 1000 ppm stock (VHG Labs, 6

Manchester, NH, USA), using the same solution used to dilute the samples. Measurement of 7 plasma zinc was performed with a Thermo XII Series ICP-MS (Thermo Electron Corporation, 8

Waltham, MA, USA), using an external calibration curve containing zinc (blank to 2000 ppb). 9

The instrument was operated with a Peltier-cooled impact bead spray chamber, single piece 10 quartz torch (1.5 mm i.d. injector) together with Xi interface cones and a Cetac-ASX 100 11 autosampler (CETAC Technologies, Omaha, Nebraska, USA). A Burgener Trace nebuliser 12 was used as this device does not block during aspiration of clinical samples. The instrument 13 was operated in standard mode (non-CCT), using 1400 W RF power, 1.10 L/min nebuliser gas 14 flow, 0.70 L/min auxiliary gas flow, 13.0 L/min cool gas flow, 70 ms dwell time, 30 s sample 15 uptake and 35 s wash time (2 repeats per sample). Zinc ion availability of PBMC was 16 measured with zinpyr-1 (from Neurobiotex, Galvestone, TX, USA) by flow cytometry, as 17 described [START_REF] Malavolta | Single and three-color flow cytometry assay for intracellular 23 zinc ion availability in human lymphocytes with Zinpyr-1 and double 24 immunofluorescence: relationship with metallothioneins[END_REF]. ). Briefly, PBMCs (1x10 6 ) were stained with 20 μM 18 Zinpyr-1 (ZP1) (Neurobiotex, Galveston, Texas, USA) for 30 min at 37C and 5% CO2 in 19 HEPES-buffered "zinc-free" RPMI Medium supplemented with 1 mM EDTA. Following 20 incubation, cells were analyzed with a flow cytometer (Coulter Epics XL, Coulter, USA). The 21 population of lymphocytes was selected using FSC and SSC and the ZP1 derived fluorescence 22 was detected at 525 nm using 488 nm exitation wavelength. Data for zinc ion availability are 23 reported as the mean fluorescence intensity normalized to the minimum fluorescence obtained 10% fetal bovine serum, 2 mM L-glutamine, 100 g/ml streptomycin and 100 IU/ml penicillin 7 (all from Gibco-Invitrogen, Carlsbad, CA, USA) and incubated for 16 hours at 37 °C in a 5% 8 CO 2 incubator at isobaric oxygen. Each PBMC sample was divided into control and heat 9 shock aliquot. Heat shock was performed at 43 °C for 1h in an Eppendorf thermomixer at a 10 cell density of 10 6 /ml. The induction of the heat shock response was allowed to develop for 4 11 h at 37 °C in a 5% CO 2 incubator. Control PBMCs of the same person were treated identically 12 except for exposing the cells to heat shock. Then cells were harvested, washed with PBS 13 supplemented with 2% fetal bovine serum, counted and the viability was assessed using a 14 CASY TT cell counter (Scharfe Systems, Bielefeld, Germany). Viability was over 90%. 15 16 After washing once with Perm/Wash solution and once with PBS with 2% FBS cells were 3 fixed with 2% paraformaldehyde in PBS and analyzed with a FACSCalibur flow cytometer 4 using the CellQuest software (BD Biosciences, San Diego, CA, USA). Approximately 5000 5 live lymphocytes from each sample were electronically gated according to granularity and size 6 in the forward versus side scatter (Figure 1A). Differentiation between CD3+ and CD3-7 subpopulations was achieved in the CD3-APC vs. side scattergram (Figure 1B). Hsp70-8 related fluorescence was obtained in a 530/30 (FITC) filter, and the relative Hsp70 protein 9 level was expressed as the mean fluorescence intensity (MFI) of FITC from logarithmic 10 histograms. As a standard, two parallel vials of lymphocytes isolated from the buffy coat of a 11 healthy subject (purchased from the Hungarian National Blood Transfusion Service, approved 12 by the National Ethical Committee, and in accordance with the Helsinki regulations) were 13 used in each experiment including heat shock treatment, staining and FACS analysis.

Intracellular staining and flow cytometry 17

Characteristics of the stress response in lymphocyte populations 2

In order to gain insight into the stress response in specific subpopulations of 3 lymphocytes, we set up a two-channel FACS analysis, using CD3 (Figure 1AB), a T-4 lymphocyte marker, and Hsp70, the major cytosolic stress protein. Initial experiments were 5 performed on pooled PBMC-s from healthy donors. While basal Hsp70 level reflects steady-6 state chaperone capacity, stress-induced Hsp70 level determines the robustness of adaptation 7 to a noxious insult. Heat shock, a model of fever was selected not only as the archetype of 8 stress but also as an important physiopathological determinant of lymphocyte function. The 9 optimal conditions of induction were found to be 43˚C for 1 hour and 4 hours of recovery time 10 to allow Hsp70 synthesis (data not shown) and used in subsequent comparative studies . 11 Looking at the Hsp70 distributions, a high and a low Hsp70-expressing population 12 could be observed, and the high population could respond more dynamically to heat shock 13 (Figure 1C all, 37 vs. 43˚C). Further examining the relationship between baseline and heat-14 induced Hsp70 levels, we investigated the stress response in PBMC obtained from old donors. 15 We found a significant correlation between these variables in the CD3+ lymphocyte 16 populations, while no such strong association existed in CD3-lymphocytes (Figure 2). 17

Moreover, the strong correlation persisted with respect to inducibility, indicating that it is 18 indeed a relationship between the basal level and the mounting of the stress response. We did 19 not observe any gender-specific effect. 20 21

Zinc supplementation boosts the stress response 22

Next, we tested the effect of clinical zinc supplementation on the stress response. [START_REF] Sőti | Molecular chaperones as regulatory elements chaperone occupancy as a potential switch[END_REF] Seven healthy elderly subjects received 10 mg zinc/day for 48 ± 2 days, and blood was 24 withdrawn before and after the supplementation. Zinc supplementation did not significantly

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M A N U S C R I P T 10 increase plasma zinc concentration, however, it promoted a significant improvement of zinc 1 availability, an intracellular measure of free zinc ion available for cellular reactions, therefore 2 a better marker of zinc homeostasis (Table 1). Figure 3 shows the individual characteristics 3 of the stress response in the total lymphocyte population before and after zinc 4 supplementation. All the basal (panel A) and heat-induced Hsp70 levels (B) as well as the 5 inducibility (C) were boosted by oral zinc supplementation, as can be seen from the upward 6 trend of the individual subjects. However, heat induction of Hsp70 showed a more uniform 7 and stronger increase. 8

Statistical analysis showed a significant increase of the heat-induced Hsp70 as well as 9 the stress-inducibility upon zinc supplementation, while the basal Hsp70 level displayed a 10 tendency to increase, probably due to the limited number of subjects (Figure 4). Zinc affected 11 the CD3-population to a smaller extent. It should be also noted that CD3-lymphocytes 12 displayed a higher basal level but a less intense heat induction of Hsp70 than CD3+ cells 13 (Figure 4). Furthermore, the lymphocyte fraction that highly express Hsp70 (gated at Hsp 70 14 MFI>18) showed a sharp increase both under basal as well as heat-induced conditions upon 15 zinc supplementation (basal: 9.6 ± 2.9% before zinc vs. 22.1 ± 6.3% after zinc, p = 0.026; 16 heat-induced: 22.0 ± 16.7% before zinc vs. 57.8 ± 5.5% after zinc, p = 0.004, respectively). 17

To further investigate the effect of orally taken zinc on the stress response, we 18 compared the Hsp70 status of subjects (Age: 76.6 ± 1.7, n = 10) after zinc supplementation 19 with that of subjects (Age: 72.2 ± 1.8, n = 10, p = 0.095 for age) not having taken zinc tablets. 20

Figure 5 demonstrates that zinc-supplemented subjects had higher Hsp70 levels and 21 inducibility in all lymphocyte populations. 22 23

Robustness of the stress response correlates with intracellular zinc 24 availability

analyze the relationship between zinc status and Hsp70 levels/inducibility. We clustered the 2 subjects according to either plasma zinc or intracellular zinc availability in low or high 3 groups. Subjects with high zinc availability displayed a higher steady state Hsp70 level and a 4 more robust stress response. Plasma zinc levels did not significantly differ between groups 5 (Table 2). Using the same subjects, further calculation of bivariate linear correlations of basal 6 Hsp70 vs. zinc availability and heat-induced Hsp70 vs. zinc availability showed that both the 7 basal as well as the heat-induced Hsp70 level correlated significantly with intracellular zinc 8 availability in a concentration-dependent manner (basal Hsp70 vs. intracellular zinc 9 availability: R2=0.3260, p=0,005; heat induced Hsp70 vs. intracellular zinc ion availability: 10 R2=0,4137, p=0,002). In contrast, plasma zinc level seemed to have no significant correlation 11 with heat induced Hsp70 levels (R2=0,0048, p=0,772). We observed no gender-specific 12 effect. 13 Stress tolerance, the adaptation to an environmental noxa depends on the robustness of 3 the stress response, i.e. the inducibility of stress proteins by the heat shock transcription factor 4 [START_REF] Voellmy | On mechanisms that control heat shock transcription factor activity in 20 metazoan cells[END_REF]. Preconditioning (exposure to a mild stress, like hormesis [Verbeke et al., 5 2001;Rattan, 2004]) results in elevations in Hsp70 and other stress proteins, a more robust 6 stress response and a better adaptation with implications in longevity. However, large 7 elevation in Hsp70 is only transient and the remaining protein level is insufficient to meet the 8 increased demand during stress. To our knowledge, no molecular mechanism has been 9 associated with a better mounting of the stress response after a previous/chronic mild stress. 10

We observed that basal Hsp70 level in the CD3+ lymphocytes strongly correlates (R 2 = 11 0.6102) with its own inducibility (Figure 2A). Our results imply that Hsp70 may indeed 12 operate a positive feedback loop chaperoning its own synthesis upon a subsequent stress, 13 providing a framework for stress tolerance. Furthermore, one can predict that cells possessing 14 higher basal Hsp70 levels might have been primed by hormetic means and have better chances 15 to adapt to stress. On the contrary, continuously very high level, such as strong overexpression 16 of Hsp70 would compromise cell physiology [START_REF] Seo | T cell lymphoma in transgenic mice expressing the human Hsp70 gene[END_REF], but this threshold either may 17 not be reached in physiological setting or in the old cells analysed in the current study. Further 18 studies are needed to explore the molecular details and its applicability as a biomarker of 19 stress tolerance. 20 Zinc is indispensable for proper immune response. Zinc deficiency is detrimental, 21 while zinc supplementation is beneficial to T-lymphocyte function [START_REF] Shankar | Zinc and immune function: the biological basis of altered 12 resistance to infection[END_REF]22 Rink and Haase, 2007). Both zinc status and immune function declines with aging (Haase et 23 al., 2006b;[START_REF] Larbi | The heat shock 20 proteins in cellular aging: is zinc the missing link?[END_REF]). Here we obtained evidence that nutritional zinc intake 24 modulates the stress response. Short term zinc supplementation markedly improved all aspects

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M A N U S C R I P T 13 of lymphocyte Hsp70 homeostasis involving also CD3-cells, in spite of their less reactivity to 1 heat shock (Figures 4). This effect was highly significant even in a relatively small study 2 population. The observations were extended and confirmed towards subjects that were or were 3 not supplemented with zinc (Figure 5). Intriguingly, plasma zinc was mostly in the normal 4 range for the subjects studied (Tables 1 and2) and there was no significant difference of 5 Hsp70 response between subjects having lower and higher than 11 M plasma zinc 6 concentration, respectively (data not shown). In contrast, intracellular zinc availability highly 7 correlated with basal and heat-induced Hsp70 levels, strongly suggesting that low zinc 8 availability exerts a negative effect on the stress response, and even without subclinical zinc 9 deficiency (plasma Zn < 11 M) zinc supplemented subjects show a more robust Hsp70 10 induction. Notably, plasma zinc concentration did not seem to affect Hsp70 inducibility. This 11 may be in part attributed to the limited number of subjects and in part to the fact that plasma is 12 often discussed not to be reliable to monitor zinc status in humans, because zinc occures in the 13 body primarily intracellularly and there is only a small portion of zinc in the plasma mainly 14 bound to plasma proteins. Furthermore, plasma zinc is highly dynamic and is profoundly 15 affected by several factors such as diurnal rhythm, stress, infection, starvation and plasma 16 protein levels [START_REF] Wood | Assessment of marginal zinc status in humans[END_REF]. Alternatively, zinc status is measured by erythrocyte membrane 17 zinc [START_REF] Bettger | Effects of copper and zinc status of rats on the 13 concentration of copper and zinc in the erythrocyte membrane[END_REF] or metallothioneins [START_REF] Sullivan | Metallothionein expression is increased in 7 monocytes and erythrocytes of young men during zinc supplementation[END_REF], proteins which are 18 responsible for the regulation of intracellular free zinc ions. Similarly to our results, Thomas 19 et al. (1992) showed that metallothionein concentrations in erythrocytes elevated significantly 20 while no changes in plasma zinc could be observed after zinc supplementation. Recent in vitro 21 assessments [START_REF] Malavolta | Single and three-color flow cytometry assay for intracellular 23 zinc ion availability in human lymphocytes with Zinpyr-1 and double 24 immunofluorescence: relationship with metallothioneins[END_REF]Haase et al., 2006a) and preliminary "in vivo" findings 22 (Mocchegiani, 2007;[START_REF] Cipriano | Polymorphisms in MT1a gene coding region are 16 associated with longevity in Italian Central female population[END_REF] suggest that intracellular zinc availability (or labile 23 zinc) of leukocytes may be a parameter that allows a good determination of zinc status. The metallothioneins what is essential to regulate many cellular processes such as apoptosis, stress 2 response or cell cycle [START_REF] Jacob | Control of zinc transfer between thionein, 18 metallothionein, and zinc proteins[END_REF][START_REF] Malavolta | Single and three-color flow cytometry assay for intracellular 23 zinc ion availability in human lymphocytes with Zinpyr-1 and double 24 immunofluorescence: relationship with metallothioneins[END_REF]. Moreover, since stress 3 signals mobilize zinc by oxidation of protein sulphur ligands (Maret, 2006), the assessment of 4 labile zinc represent an optimal target for establishing a link between stress response and zinc 5 status. In addition to these, this method allowed also the determination of zinc in the cells 6 analyzed while plasma zinc is not a cell specific indicator of zinc status. Our findings 7 corroborates in vitro experiments that reported a stress response inducing property of zinc in 8 various cell cultures and peripheral cells of different origin [START_REF] Hatayama | Regulation of hsp70 6 synthesis induced by cupric sulfate and zinc sulfate in thermotolerant HeLa cells[END_REF]Ambra et 9 al., 2004;[START_REF] Bauman | Production of metallothionein and heat-shock 11 proteins in response to metals[END_REF]Odashima et al., 2002;Quing et al., 2004 and reviewed in 10 Arslan et al., 2006). Similarly, zinc in our study also increased the basal steady-state Hsp70 11 level in human lymphocytes, suggesting that zinc per se exerts a mild stress on a variety of 12 cell types. This property of zinc might resembles an hormetic action (Rattan, 2004). In fact, 13 the zinc-induced increase in Hsp70 elicited a more intense stress response (2-4-fold increase 14 of heat-induced Hsp70) with no apparent damage. Moreover, PBMC from zinc-supplemented 15 people had much better morphology (data not shown), arguing for a generally improved 16 ability to cope with stress. Since blood samples were obtained and frozen under controlled 17 conditions at different times by the same personnel, the effect observed was not due to 18 differential sample preparation. 19 Chaperones and zinc may co-operate in other ways, as well. Besides, zinc is also an 20 important regulatory metal in protein function and signaling. Both zinc and chaperones 21 promote lymphocyte proliferation [START_REF] Csermely | Zinc can increase the activity of 19 protein kinase C and contributes to its binding to plasma membranes in T lymphocytes[END_REF][START_REF] Huse | A Zn2+ ion links the cytoplasmic tail of CD4 and 16 the N-terminal region of Lck[END_REF]Schnaider et al., 22 2000). Lack of zinc results in decreased cell proliferation, skin and mucous membrane 23 manifestations and decreased wound healing (Prasad, 2003), while chaperone upregulation 24 facilitates wound healing and regeneration [START_REF] Vígh | 17 Bimoclomol: a nontoxic, hydroxylamine derivative with stress protein-inducing activity 18 and cytoprotective effects[END_REF]. Moreover, zinc deprivation zinc deprivation and during in vitro aging, suggesting a compensatory mechanism. As HSF-1 3 is a zinc finger protein, it may be directly affected by fluctuations in intracellular free zinc ion 4 availability. This is supported by the fact that free intracellular zinc is in the femtomolar 5 range, around the K D of various zinc-binding proteins (Outten et al, 2001) and p53 and the 6 apurinic/apyrimidinic endonuclease are all inactivated by zinc deficiency (Ho and Ames, 7 2002;[START_REF] Ho | Zinc deficiency induces oxidative DNA damage 12 and increases p53 expression in human lung fibroblasts[END_REF]. According to a recent intriguing hypothesis of Bruce Ames (Ames, 8 2006), shortage of micronutrients may promote the re-allocation of scarce micronutrients 9 towards processes favoring short term survival at the expense of longevity. According to this, 10 the stress response (a major adaptation mechanism) would either enjoy an intermediate 11 priority (after basic cellular processes like oxidative metabolism, and before reproduction) and 12 as such, would be downregulated during modest nutrient deprivation, as seen in the present 13 study. It is very much in agreement with the fundamental role of the stress response in 14 longevity [START_REF] Sőti | Aging and molecular chaperones[END_REF]. Therefore, beyond genomic stability, the stress response 15 is another candidate mechanism to connect nutrient scarcity with aging and degenerative 16 diseases, at least in the cases of zinc. Effect of other micronutrients on the stress response 17 remains to be elucidated. 18 As a conclusion, we provided evidence that dietary zinc is a chaperone inducer and a 19 major determinant of the stress response in human lymphocytes in the elderly. Since the stress 20 response is a conserved and universal phenomenon, and the effect of zinc in vitro is fairly 21 general, these findings may have wider implications in other bodily tissues as a possible anti-22 aging mechanism. However, whether this is a proof of principle or a special exception will be 23 a subject of future studies.

A 1 ( 4 2. 4 .

 144 C C E P T E D M A N U S C R I P T 7 after the addition of 50μM N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) Sigma-Aldrich, MI, Italy). 2 3 Cell culture and heat shock treatment 5 PBMC were gently thawed in RPMI 1640 medium, supplemented with 10 mM Hepes, 6

  Cells were fixed and permeabilized in 250 μl Cytofix/Cytoperm solution (BD 18 Biosciences Pharmingen, San Diego, CA, USA) at 4 °C for 20 minutes. Cells were then 19 washed two times with Perm/Wash solution (BD Biosciences Pharmingen, San Diego, CA, 20 USA) and incubated with flourochrome-conjugated mouse monoclonal antibodies [anti-CD3 21 allophycocyanin (APC) conjugated antibody (345767, BD Biosciences, San Diego, CA, USA) 22 and anti-Hsp70 fluorescein isothiocyanate (FITC) conjugated antibody (SPA-810FI, 23 Stressgen, Alberta, Canada)] at 4 °C for 30 minutes. (CD3 immunostaining either in intact A C C E P T E D M A N U S C R I P T 8 were stained either with anti-CD3-APC conjugated antibody or with anti-CD3 FITC 1 conjugated antibody (345763, BD Biosciences, San Diego, CA, USA) as described above. 2

14

 14

15 2. 6 .

 156 Statistical analysis 16 Data were statistically analyzed using Statistical Package for the Social Sciences 17 software version 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Variables are expressed as 18 mean ± standard error of the mean (SEM). Means were either compared by using the 19 Student's paired samples or independent samples t-test. Correlation of continuous variables 20 was analyzed by the Spearman algorithm. A p value (two-tailed) < 0.05 was considered 21 statistically significant. A C C E P T E D M A N U S C R I P T 9 3. Results 1

14 4 .

 4 Discussion 15 Loss of adaptation to stress is a hallmark of aging. A major molecular mechanism 16 behind is a decrease in heat shock response, a fundamental cytoprotective and survival 17 mechanism in several models[START_REF] Sőti | Aging and molecular chaperones[END_REF][START_REF] Arslan | Protein homeostasis and molecular chaperones in 9 aging[END_REF], including human 18 blood cells and extracellular chaperones of the elderly (Njemini et al., 2002; Rea et al., 2001; 19 Singh et al., 2006). Since molecular chaperones are intimately involved in signaling and 20 proliferation, a less robust stress response may not only cause limited survival, but may also 21 contribute to the compromised reactivity of old lymphocytes to various inflammatory stimuli, 22 a phenomenon called immune cell anergy (Pawelec, 2006). Besides, looking at two 23 lymphocyte subpopulations it turned out that CD3-cells had a significantly higher basal and a 24 lower heat-induced Hsp70 level then their CD3+ counterparts. Whether the CD3-B-cells (and subjects and if it relates to immune function is an intriguing open question. 2

A

  C C E P T E D M A N U S C R I P T 14 of zinc which is modulated by the release of zinc from intracellular vesicular stores or from 1

A

  C C E P T E D M A N U S C R I P T 15 compromises the stress response in ketratinocytes, and in the Jurkat T-cell line (Larbi et al., 1 2006; Parat et al., 1998). An upregulation of HSF-1 was observed in T-cell clones both upon 2

1 17 Footnote 1 21Figure legends 1 2 Figure 1 9 Figure 2 17 Figure 3 Figure 4 11 Figure 5 22 AFigure 5 of

 117121921734115225 Figure legends1

2.3. Plasma zinc and intracellular zinc ion availability determination

non-permeabilized or in permeabilized cells gave identical results.) For compensation cells

intracellular zinc that is available for zinc-dependent enzymes is often called as the labile pool

(abstr).

of cellular networks. Curr. Opin. Cell Biol. 17, 210-215.

Student's independent samples t-test.

 2Relationship between the zinc status and the stress response. Subjects 10 were grouped according to their zinc availability (high Zn > 1.30 ≥ low Zn). Data were 11 obtained as described in Subjects and methods and shown as mean ± SEM. Basal (37˚C) and 12 heat-induced Hsp70 (43˚C) levels are indicated as the mean fluorescence intensity (MFI), 13 Hsp70 inducibility (Ind.) is given as the 43˚C/37˚C ratio. p values were calculated using the


a recovering beam search algorithm

Keywords: Scheduling, single-machine, common due date, earliness-tardiness penalties

Minimizing earliness-tardiness penalties for common due date single-machine scheduling problems by

Introduction

In recent decades, production scheduling requirements for meeting a common due date have been investigated extensively, especially since the just-in-time (JIT) philosophy was widely applied in different industries. In general, common due date problems can be classified into two categories: restrictive and unrestrictive ones. If the optimal value of a common due date has to be determined or is given and has no influence on the optimal sequence of jobs, it is called unrestrictive. On the other hand, if a common due date is given and may influence the optimal sequence of the jobs, it is called restrictive. Therefore, a search for an optimal sequence of the jobs has to be carried out with respect to the due date [START_REF] Feldmann | Single-machine scheduling for minimizing earliness and tardiness penalties by meta-heuristic approaches[END_REF].

Meeting a restrictive common due date is a general scheduling issue in practice.

Such instances might occur if a customer orders different variations of a product, all of which must be delivered in the same truck or container ship for transportation cost saving [START_REF] Sule | Industrial scheduling[END_REF], or if a firm has installed a weekly bulk delivery to the wholesaler [START_REF] Feldmann | Single-machine scheduling for minimizing earliness and tardiness penalties by meta-heuristic approaches[END_REF]. A survey of US manufacturing practitioners reveals that meeting a restrictive common due date is one of the most important scheduling criteria [START_REF] Wisner | A survey of US manufacturing practices in make-to-order machine shops[END_REF]. When scheduling a production system, jobs completed before the common due date incur earliness penalties, which may include holding costs for finished goods, deterioration of perishable goods and opportunity cost. On the contrary, jobs completed after the common due date sustain tardiness penalties, which may include loss of future sales and rush shipping cost. Besides, the importance of a customer can also be taken into account by using different weights for orders [START_REF] Dauzère-Pérès | An exact method to minimize the number of tardy jobs in single machine scheduling[END_REF]. Hence, in connection with JIT production and delivery, earliness and tardiness penalties are of continuing interest for scheduling researchers and practitioners. However, scheduling problems with a restrictive common

A C C E P T E D

M A N U S C R I P T 4 due date where the objective is to minimize earliness-tardiness penalties is not a trivial issue. Even a relatively simple problem, such as the class of a single-machine, is NP-hard [START_REF] Hall | Earliness-tardiness scheduling problems[END_REF].

This study focuses on restrictive common due date single-machine scheduling problems with respect to total weighted earliness-tardiness penalties. In the standard three-field notation this problem is denoted as 1/ / ( )

res i i i i i d d E T α β = +
. Due to the nature of the complexity of this problem, global optimal solutions cannot be easily obtained when the problem size is large. Thus, for the 1/ / ( )

res i i i i i d d E T α β = +
problem, no efficient exact methods are known to exist when no restrictions are placed on the values of the penalty per unit time of earliness ( i α ) and the penalty per unit time of tardiness ( i β ).

Despite this discouraging theoretical result, the past three decades have witnessed growing interest in the 1/ / ( )

res i i i i i d d E T α β = + problem. A comprehensive survey
on relevant studies of this subject was provided by [START_REF] Gordon | A survey of the state-of-art of common due date assignment and scheduling research[END_REF]. Researchers developed polynomially or pseudo-polynomially solvable algorithms for special cases where conditions for i α and i β are imposed (Cheng & Gupta, 1989; Hall et al., 1991; Hoogeveen & van de Velde, 1991; Kahlbacher, 1993). Studies employing meta-heuristics [START_REF] Lee | Parallel genetic algorithms for the earliness-tardiness job scheduling problem with general penalty weights[END_REF][START_REF] James | Using tabu search to solve the common due date early/tardy machine scheduling problem[END_REF][START_REF] Feldmann | Single-machine scheduling for minimizing earliness and tardiness penalties by meta-heuristic approaches[END_REF][START_REF] Hino | Minimizing earliness and tardiness penalties in a single-machine problem with a common due date[END_REF] and dealing with this argument are even more recent. These recent published meta-heuristics do provide excellent results, but in some cases these meta-heuristics are so intricate that independent coding is unlikely to lead to the same effectiveness or efficiency.

The recovering beam search (RBS) algorithm, first proposed by Della Croce and T'kindt (2002), is one of the most recent and hopeful hybrid heuristics for combinatorial

A C C E P T E D M A N U S C R I P T 5
optimization problems. The RBS algorithm is an enhancement method of beam search (BS), which is a truncated branch and bound algorithm for solving combinatorial optimization problems. In the BS search procedure, only the w most promising nodes at each level of the search tree are retained for further branching, instead of all nodes, and w is the so-called beam width. Meanwhile, the remaining nodes are pruned off permanently. Since only w nodes are retained at each level of the search tree, the running time of BS is polynomial in terms of the problem size [START_REF] Valente | Filtered and recovering beam search algorithms for the early/tardy scheduling problem with no idle time[END_REF].

However, if a node leading to the optimal solution is discarded during the search process, there is no way to later reach the optimal solution using the classic BS. The RBS algorithm seeks to improve upon previous possibly incorrect decisions through the use of a recovering phase that searches for improved partial solutions dominating those selected by the beam. Notably, to guarantee that the total number of explored nodes is polynomial, a partial solution may be only substituted by another partial solution with the same depth level of the search tree in the recovering phase [START_REF] Della Croce | A Recovering Beam Search algorithm for the one-machine dynamic total completion time scheduling problem[END_REF].

A number of successful applications of the RBS method on scheduling problems [START_REF] Della Croce | Recovering Beam Search: Enhancing the beam search approach for combinatorial optimization problems[END_REF][START_REF] Ghirardi | Makespan minimization for scheduling unrelated parallel machines: A recovering beam search approach[END_REF][START_REF] Valente | Filtered and recovering beam search algorithms for the early/tardy scheduling problem with no idle time[END_REF][START_REF] Esteve | A recovering beam search algorithm for the single machine Just-In-Time scheduling problem[END_REF] have recently appeared in the literature. In an attempt to assist in reducing the gap between theory and practice, this study proposes an RBS algorithm for the 1/ / ( )

res i i i i i d d E T α β = +
problem. The remainder of this paper is organized as follows. In the next section, the problem is formulated. Section 3 gives a detailed description of the proposed RBS algorithm. The computational results of applying the proposed RBS algorithm to a famous benchmark problem set are provided in section 4, as well as comparisons of the performances with nine meta-heuristics from the relevant literature. Finally, this study concludes with suggestions for possible future research.

A C C E P T E D M A N U S C R I P T 6

Problem formulation

The 1/ / ( )

res i i i i i d d E T α β = +
problem can be defined as follows. Consider a set of n jobs, each of which requires exactly one operation to be scheduled on a single-machine with a restrictive common due date d . For each job i , the processing time i p , the penalty per unit time of earliness i α , and the penalty per unit time of tardiness i β are given in advance.

A

n i i i i i E T α β = +
. Besides these parameters, the following assumptions are made:

Every job is processed without preemption.

The setup times of the jobs are included in the processing times and are sequence-independent.

The ready time of each job is zero; namely all the jobs are available at the beginning of the scheduling period.

The machine can process no more than one job at a time and is continuously available.

The proposed RBS algorithm for the 1 / / ( )

res i i i i i d d E T α β α β α β α β = + = + = + = + problem
For a common due date case with general earliness-tardiness penalties, there is an optimal solution satisfying the following three optimality properties. To obtain the

A C C E P T E D M A N U S C R I P T 7
objective function value more efficiently, the following section develops and elaborates on the proposed RBS algorithm by using the three well-established properties.

Property 1. An optimal schedule does not contain any idle time between any consecutive jobs [START_REF] Kahlbacher | Scheduling with monotonous earliness and tardiness penalties[END_REF].

Property 2. The optimal schedule is V-shaped around the common due date. In the optimal schedule, the jobs completed before or on the common due date d are scheduled in a non-increasing order of the ratios i i p α , and the jobs starting on or after d are scheduled in a non-decreasing order of the ratios i i p β (Smith, 1956).

Property 3. In the optimal schedule, either the first job starts from time zero or the completion time of one job coincides with the common due date d [START_REF] Hoogeveen | Scheduling around a small common due date[END_REF].

Consider a 1/ / ( )

res i i i i i d d E T α β = +
problem with depth size n (i.e. n jobs)

of the search tree. A flowchart of the RBS algorithm is depicted in Figure 1. The following subsection will discuss the related steps in detail.

| Insert Figure 1 about here | → ←

Step 1: Initialization

Set 0 l = and 1 { } S σ =
, where l denotes the search tree level, S represents the vector of the best current partial solutions at a given level, and 1 σ be the parent (root) node.

Step 2: Main loop A. For each best current partial solution k σ in S , branch k σ to generate the corresponding child nodes. (b) Update the vector of the best current partial solutions S by the w partial solution found so far.

Step 3: Stopping criterion:

Let 1 l l = + , IF l n < : GO TO step 2; ELSE STOP: rearrange each partial solution in S according to properties 1~3 and find the best solution.

The SEA priority evaluation function

A C C E P T E D M A N U S C R I P T 9
Recently, we proposed a quick method, namely the sequential exchange approach (SEA; [START_REF] Lin | A sequential exchange approach for minimizing earliness-tardiness penalties of single-machine scheduling with a common due date[END_REF], to obtain a near optimum solution for 1/ / ( )

res i i i i i d d E T α β = +
problems. This simple heuristic firstly divides the jobs into two sets: E S and L S , where jobs in set E S start processing before the common due date, and jobs in set L S start processing on or after the common due date. Then, SEA swaps or moves jobs between the two sets systematically to derive a near optimum solution.

The priority evaluation function of the filter procedure at each search tree level is a key issue in the RBS technique. In this study, a computationally inexpensive filtering procedure, namely the SEA priority evaluation function is applied to select some of the children of each beam node for a more detailed evaluation. Assume that a near optimum solution π is obtained by the SEA. Based on the job j 's order number ([ ]

j ) in E S
or L S of solution π , we set the priority evaluation function value of job (node) j

( j PEFV ) as follows: [ ] 1, if job [ ], if job E j L n j j S PEFV j j S -+ ∈ = ∈
Then, the ϕ filtered child nodes with the best SEA priority evaluation function values are added to set C for further evaluation.

The evaluation function

After being filtered by the SEA priority evaluation function, the ϕ selected child nodes are then accurately evaluated via a function V defined by (1 ) +

V LB UB γ γ = - ,
where γ is a given weight and LB and UB refer to a lower bound and an upper bound, respectively.

A C C E P T E D M A N U S C R I P T

10

In this study, we propose the following heuristic to obtain UB of the partial schedule ' π represented by the node. The idea for this heuristic is based on work completed by [START_REF] Dileepan | Common due date scheduling problem with separate earliness and tardiness penalties[END_REF] and [START_REF] Biskup | Benchmarks for scheduling on a single machine against restrictive and unrestrictive common due dates[END_REF].

Heuristic of UB.

Let A S and B S be the ordered sets of jobs that are started after the common due date and completed before the common due date, respectively. The main steps of the UB heuristic are as follows.

Step 1: Sequentially assign the jobs of the partial schedule ' π into B S until the time gap

B gap i i S T d p ∈ = -
is smaller than the assigned job's processing time. Then, add the remaining jobs of ' π to set A S in a non-decreasing order of i i p β .

Step 2: IF A S = ∅ : GO TO step 3; ELSE: GO TO step 5.

Step 3: Add to set P αβ all the jobs which are not included in ' π according to the decreasing ratios i i β α . The tiebreak rule gives preference to the job with the smallest value of i β .

Step

n i i i i i Z E T α β = = + s.t. , 1, 2, ..., , i i i i T s p d E i n = + -+ = (1)
(1 ), 1, 2, ..., 1; 1, ..., ,

i i k ik s p s M x i n k i n + ≤ + - = - = + (2) , 1, 2, ..., 1; 1, ..., , k k i ik s p s Mx i n k i n + ≤ + = - = + (3)
, , 0, 1, 2, ..., ,

i i i T E s i n ≥ = (4) {0, 1}, 1, 2, ..., 1; 1, ..., . ik x i n k i n ∈ = - = + (5)
The values for earliness and tardiness are calculated by constraint (1). Constraints

(2) and (3) determine the starting times of the jobs. If job i is sequenced prior to job A Lagrangian relaxation of constraint (1) yields the Lagrangian subproblem (LR): If

A C C E P T E D M A N U S C
(LR) [ ] 1 ( ) min ( ) ( ) ( ) s.t. (2) (5), n i i i i i i i i i i L s p d E T λ λ α λ β λ = = + -+ + + - - where ( ) 1
0 i i α λ + < or 0 i i β λ -< for some i ( 1, 2, ..., i n = ), then we have i E = ∞ or i T = ∞ , giving ( ) L λ = -∞ .
To avoid this, we therefore require that

i i i α λ β -≤ ≤ for each i ( 1, 2, ..., i n =
). The Lagrangian subproblem (LR) is solved by setting

0 i i E T = = for each i , where 1, 2, ..., i n =
, and then scheduling the jobs in a non-increasing order of / i i p λ according to Smith's rule [START_REF] Smith | Various optimizers for single-stage production[END_REF].

In general, the Lagrangian multipliers can be iteratively updated using the subgradient optimization method (SOM) to improve the lower bound. However, experiments have shown that it takes a lot of time to get the SOM to converge [START_REF] Sourd | The one-machine problem with earliness and tardiness penalties[END_REF]. The speed increases of the multiplier adjustment method (MAM) more than sufficiently compensate for the possible loss in lower quality over the SOM, we resort to using the MAM.

The MAM first requires a heuristic to sequence the jobs, and then chooses the multipliers so that the resulting lower bound is as large as possible. Let i s * be the start time of job i ( 1, ..., i n =

) in the job sequence generated by the heuristic. To obtain the maximum value of LR, we solve the problem LR( i s * ):

A C C E P T E D M A N U S C R I P T 13 W = 1 max ( ) n i i i i s p d λ * = + - (LR( i s * )) s.t. 1 1 / / , i i i i p p i λ λ + + ≥ ∈early jobs, (6) 1 1 / / , i i i i p p i λ λ + + ≤ ∈tardy jobs, (7)
* * , 1, 2, ..., .

i i i i n α λ β -≤ ≤ = (8)
where * max{ / , , 1, ..., }

i i k k p p k i i n α α -= - = + and * min{ / , 1, 2, ..., } i i k k p p k i β β = = .
To obtain the Lagrangian multipliers more rapidly, we use the weighted shortest processing time (WSPT) and the weighted longest processing time (WLPT) to generate the initial job sequence and i s * . Then, we calculate

* i i i R s p d = + -( 1, 2 ,..., i n = ),
and set the Lagrangian multipliers as follows:

* , if 0, i i i R β = ≥ * , if 0. i i i R α = - <
After obtaining the Lagrangian multipliers, we can get the solution to the problem LR( i s * ) and set it as the LB of the evaluation function V .

Computational results and discussion

Test problems

One difficulty faced by researchers in scheduling is to compare their developed heuristics with those of other researchers. If a standard set of test problems is accessible, different algorithms' performances can be compared on exactly the same set of test problems. For this reason we chose 280 benchmark problems from [START_REF] Biskup | Benchmarks for scheduling on a single machine against restrictive and unrestrictive common due dates[END_REF] as the test problems for this study.

Biskup and Feldmann have generated benchmark problems with the number of jobs n = 10, 20, 50, 100, 200, 500 and 1000 for the 1/ / ( )

res i i i i i d d E T α β = + A C C E P T E D M A N U S C R I P T 14
problem. The restrictive factor h for determining the common due date d , which can be computed as

i d h p =
, was set to be 0.2, 0.4, 0.6 and 0.8. For each combination of n and h , ten instances were generated, respectively. Thus, the test bed comprised 280 problems.

Computational results

The choice of parameters may affect the quality of the results. Choosing appropriate values for the parameters is time consuming and in general seems dependent on the set of data. A number of preliminary experiments were performed to fine tune the parameters of the proposed RBS algorithm. The experiments conducted here reveal that the filter width ϕ and the beam width w yielded better quality within reasonable computational expenses at values of around 6 and 3, respectively. The experiments also indicate that the parameter γ yielded better quality at values of around 0.3, 0.4, 0.5, and 0.6 for h = 0.2, 0.4, 0.6 and 0.8 problems, respectively. Consequently, these values were used for the experiments conducted in this study. [START_REF] Hino | Minimizing earliness and tardiness penalties in a single-machine problem with a common due date[END_REF] proposed four meta-heuristics, including tabu search (TS), genetic algorithm (GA), hybrid of tabu search and genetic algorithm (HTG), and hybrid of genetic algorithm and tabu search (HGT) to the same benchmark problems. Each instance was solved over ten trials by each of these four meta-heuristics and the best solutions obtained were recorded.

| Insert Table 1 about here | → ←

To compare the effectiveness of the different approaches, the solution quality is measured by listing the average improvement rate (%) of the objective function values from the upper bounds proposed by [START_REF] Biskup | Benchmarks for scheduling on a single machine against restrictive and unrestrictive common due dates[END_REF]. As revealed in Table 1, on the whole, the proposed RBS algorithm outperformed the nine meta-heuristics.

Total average improvement rates of 6.72 and 4.37 for the problems with the restrictive factors h = 0.2, 0.4, and h = 0.2, 0.4, 0.6, 0.8 were achieved, respectively. The proposed RBS algorithm was superior in all of the different sized problems with different values for the restrictive factor. Notably, for all of the n = 10 instances, the optimal solutions (determined by applying LINGO to their integer programming models) were obtained by the proposed RBS heuristic, too. These analytical results

clearly indicate that the proposed algorithm is a promising approach for solving the 1/ / ( ) As revealed in Table 2, the computation time required by the proposed algorithm is very short. Since the computation times vary with hardware, software and coding, this study did not directly compare computational efficiency. Nonetheless, even for problems involving up to 1000 jobs, the proposed RBS algorithm can obtain very good solutions within reasonable computational expenses. This indicates that the proposed RBS heuristic can be applied to real world problems.

res i i i i i d d E T α β = + problem.
On the other hand, as the running time of the meta-heuristics proposed by [START_REF] Hino | Minimizing earliness and tardiness penalties in a single-machine problem with a common due date[END_REF] is provided as a graph, therefore, only approximate values of the GA and the hybrid meta-heuristics (i.e. HTG and HGT) can be used for comparison. The average running time of n = 1000 is over 90 and 32 seconds of the GA and the hybrid meta-heuristics on a Pentium 4 (1.7GHz) PC, respectively. Notably, the best objective function values of these meta-heuristics were obtained from ten runs while the objective function values of the proposed RBS algorithm were obtained from one trial. These results indicated that the proposed RBS algorithm is a more efficient and effective approach compared with these state-of-the-art meta-heuristics.

Conclusions

Single-machine is one of the most useful models in practice, since a complex system can be reduced to a single-machine problem, especially if there is a bottleneck machine in the system. The purpose of this study is to make a step towards establishing an effective heuristic for the 1/ / ( )

res

  Add to C the ϕ child nodes that are selected by the SEA priority evaluation function, where C denotes a set of filtered offspring nodes and ϕ is a filter width parameter defined by experimental testing. (b) For each child node in C , compute its evaluation function value (denotes the upper bound weight in the weighted sum of a lower bound ( LB ) and an upper bound (UB ) on the optimal solution value of that partial solution. (c) Sort the child nodes in C in a non-decreasing order of their evaluation function values and add to T the w child nodes with the best evaluation function values, where T denotes a set of beam nodes and w is a beam width parameter defined by experimental testing. C. Recovering phase: (a) For each partial solution represented by the node in T , pick the last job in the job list and find the best sequence by placing that job in all possible positions in the partial solution.

  15 runs of the five different meta-heuristics (FDM).

  Iteratively assign the first job of P αβ to set B S and delete it from P αβ

	B.	n	2	jobs have been assigned to set B S . In this case iteratively assign
		the first of the remaining jobs of P αβ to set B S until the objective
		function value is increased. Terminate the heuristic and the current
	objective function value is saved as UB . Step 5: Shift the jobs in B S and A S to make the completion time of the final job in B S and the start time of the initial job in A S equal to the common due date d . Terminate the heuristic and the current objective function value is saved as UB . To get LB of the evaluation function , V we formulate the / ( ) res i i i i d d i E T α β = + problem, and decompose the problem into a subproblem based on the Lagrangian relaxation, and further develop an efficient multiplier 1/ adjustment method to compute the values of the Lagrangian multipliers. The M A N U S C R I P T 1/ / ( ) res i i i i i d d E T α β = + problem can be logically formulated as an integer
	until one of the following situations occurs: A. The time gap gap T is smaller than the assigned job's processing time. If in this case 0 gap T > , sequentially search for another job of P αβ which fits into the gap. As soon as 0 gap T = or no more jobs of P αβ fit into the gap, assign the remaining jobs of P αβ to set A S . Finally, sequence all jobs in A S and B S according to property 2 (the programming problem (P): 1 A C C E P T E D min ( )
		V-shaped property).
				11

4:

  The data listed in Table 1 are the best objective function values

	The proposed RBS algorithm was coded in Visual C#.net, and run on a PC with an
	Intel Pentium 4 (2.4GHz) CPU. The final results of the proposed RBS algorithm
	compared with nine meta-heuristics proposed by Feldmann and Biskup (2003) and Hino
	et al. (2005) are shown in Table 1. Feldmann and Biskup investigated the same
	benchmark problems by applying five different meta-heuristics, namely evolutionary
	strategy (ES), ES with a destabilization phase (ESD), simulated annealing (SA),
	threshold accepting (TA) and TA with a back step (TAR). However, they only
	considered the problems with the restrictive factor h set as 0.2 and 0.4. Each
	meta-heuristics was performed three runs on each single instance with different initial
	random numbers.

Table 2 .

 2 It should be noted that the FDM proposed by Feldmann and Biskup would in general require multiple trials with different random seeds to get a better solution.

	The average computation time (CPU time in seconds) of the proposed RBS
	algorithm run on a Pentium 4 (2.4GHz) PC and that of the five different meta-heuristics
	(FDM) proposed by Feldmann and Biskup (2003) run on a Pentium 90 PC are presented
	in

Table 1 .

 1 In this study, we propose an RBS algorithm for this problem. The experimental results clearly indicate that the proposed RBS algorithm is a state-of-the-art method for solving this problem, Average improvement rate (%) of meta-heuristics and the proposed RBS algorithm from upper bounds

	i d d =	α	i i E	+	β	i i T	problem.

Figure 1. Flowchart of the RBS algorithm

Acknowledgements

The authors would like to thank Biskup and Feldmann [19] for their benchmark problem sets and solution files. This paper was financial support in part by the National Science Council of the Republic of China, Taiwan, under the Contract No. NSC 96-2221-E-211-009.

A C C E P T E D

M A N U S C R I P T 17 as seen by comparing the obtained results to the best available meta-heuristics on a wide range of benchmark instances. Further, the experimental results also indicate that the proposed approach can be applied to efficiently schedule common due-date problems with general earliness-tardiness penalties of a realistic size.

Possible directions for future research include hybridizing the RBS algorithm with meta-heuristics, such as simulated annealing (SA), genetic algorithm (GA), tabu search (TS), and ant colony optimization (ACO), and also by extending the proposed RBS algorithm to assist in solving multi-machine scheduling problems with general or non-linear earliness-tardiness penalties in regards to a common due date.

A C C E P T E D M A N U S C R I P T

22


L White

R Costarelli

E Giacconi

C Muti

J R Cipriano

E Powell

Mocchegiani

Dr Dawn J Mazzatti
email: dawn.mazzatti@unilever.com

M Malavolta

A J White

L Costarelli

R Giacconi

E Muti

C Cipriano

J R Powell

E Mocchegiani

Muti

Effects of interleukin-6 -174C/G and metallothionein 1A +647A/C single-nucleotide polymorphisms on zinc-regulated gene expression in ageing

Keywords: 6, genetics, single nucleotide polymorphism, nutritional genomics, 25 zinc, ageing, inflammation, metallothionein

Effects of interleukin-6 -174C/G and metallothionein 1A

+647A/C single-nucleotide polymorphisms on zinc-regulated gene expression in ageing

In human ageing, zinc bioavailability is diminished resulting in impaired immune 48 response and anti-oxidant activity [START_REF] Mocchegiani | Zinc, T-cell 538 pathways, aging: role of metallothioneins[END_REF]. Metallothioneins (MTs) 49 play an important role in regulating zinc homeostasis due to their high affinity for 50 zinc.

An important function of MTs is in releasing zinc in response to 51 oxidative/nitrosative stress in order to up-regulate the zinc-dependent antioxidant 52 enzymes (Mocchegiani et al. 2006). This response is inadequate in states of chronic 53 inflammation and in ageing individuals [START_REF] Mocchegiani | Metallothioneins/PARP-540 1/IL-6 interplay on natural killer cell activity in elderly: parallelism with[END_REF]. deficiency in diabetic patients (Giacconi et al. 2005). Metallothionein induction and 62 expression are under the control of IL-6 [START_REF] Jacob | Induction of metallothionein by 502 stress and its molecular mechanisms[END_REF]. These data suggest that 63 in the presence of heightened IL-6 production, the MT1a +647 C/A transition may 64 impact negatively on inflammation through up-regulation of IL-6 pro-inflammatory 65 cytokine production, thereby creating vicious pro-inflammatory feed-back cycle.

66

Since increased IL-6 production has been linked to disease and is a poor indicator of 67 survival (Fagiolo et al. 1993, Ferrucci et al 1999), these studies have profound 68 implications for understanding the link between genes, health and longevity. ). Therefore, genetic variability at the IL6 -174 C/G locus may be useful for 96 both screening of elderly subjects who are at risk for zinc deficiency and impaired 97 immune response and also in identifying subjects that are more or less able to regulate 98 zinc homeostasis (Mocchegiani et al 2006).

Human Subjects and Sample Collection

Sixteen elderly subjects (age range 69-79 years) were selected for study participation.

103

Elderly subjects were recruited in accordance to immunogeronotological 'SENIEUR' 104 protocol [START_REF] Ligthart | The SENIEUR protocol after 16 years: the next step is to 511 study the interaction of ageing and disease[END_REF]. 15 ml whole blood was collected and peripheral blood The intracellular release of zinc from lymphocytes and monocytes and total 118 metallothionein protein expression in PBMC were assessed by flow cytometry as 119 previously described [START_REF] Cipriano | Polymorphisms in MT1a 466 gene coding region are associated with longevity in Italian Central female 467 population. Biogerontology 7, 357-365. insulin receptor binding in rat adipocytes[END_REF], Malavolta et al. 2006). Zinc ion

A C C E P T E D M A N U S C

Metallothionein protein expression

128

Relative cellular fluorescence, reflecting cellular MT levels, was determined using 129 flow cytometry as described previously [START_REF] Malavolta | Single and three-color flow 520 cytometry assay for intracellualr zinc ion availability in human lymphocytes with 521 Zinpyr-1 and double immunofluorescence: relationship with metallothioneins[END_REF][START_REF] Yurkow | Flow cytometric determination of 563 metallothionein levels in human peripheral blood lymphocytes: utility in 564 environmental exposure assessment[END_REF]. Amersham, UK) according to manufacturer's guidelines using a Biorobotics TAS 179 microarray system. Probe labelling was performed using the Genisphere 350HS In the current study, we investigated whether variability in IL-6 -174 and MT1a +647 have the greatest susceptibility to nutritional deficiencies will aid in selecting 345 individuals who will be afforded the greatest health benefits and will additionally 346 provide the greatest probability of a successful nutritional intervention trial. In addition to associations between zinc status and obesity, an interaction between 364 dietary zinc and adiposity was implicated in MT-null mice [START_REF] Beattie | Obesity and hyperleptinemia in 450 metallothionein (-I and II) null mice[END_REF]. MT-365 deficient animals are obese, have elevated plasma leptin concomitant with adipose 366 leptin mRNA expression compared to controls, and pronounced alterations in zinc 367 homeostasis, suggesting that disrupted zinc availability may lead to alterations in 368 energy regulation [START_REF] Beattie | Obesity and hyperleptinemia in 450 metallothionein (-I and II) null mice[END_REF]. Furthermore, the obesity associated with these demonstrated in zinc-deprived rats [START_REF] Blanchard | Modulation of 455 intestinal gene expression by dietary zinc status: Effectiveness of cDNA arrays for 456 expression profiling of a single nutrient deficiency[END_REF], suggesting this is a 386 conserved effect that is observed in vivo. FABPs constitute a family of cytosolic lipid 387 chaperones that share a conserved structure but exhibit a distinct, tissue-specific 388 expression pattern [START_REF] Haunerland | Fatty acid-binding proteins; insights from 495 genetic manipulationis[END_REF]. FABP4 (or A-FABP) is 389 expressed predominantly in adipocytes and macrophages [START_REF] Hotamisligil | Uncoupling of obesity 499 from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid 500 binding protein[END_REF].

390

Recent studies have demonstrated that FABP4 is important in systemic metabolic 391 homeostasis. FABP4-deficient mice are partially protected against obesity-induced 392 insulin resistance, type 2 diabetes, and hypercholesterolemic atherosclerosis in both 393 dietary and genetic models (Makowski andHotasmisligil 2004, Uysal KT et al. 1997).

394

One elegant recent study performed by this same group examined the effect of leptin 395 on FABP4/5 -/-mice [START_REF] Cao | Regulation of metabolic responses by 463 adipocyte/macrophage fatty acid-binding proteins in leptin-deficient mice[END_REF]). In the absence of leptin and despite severe . 2002. , Gao et al. 2002)). These observations further establish the relationship 406 between cellular stress responses, inflammatory cytokine production and obesity.

407

Firstly, FABP4 may co-ordinate the lipid-mediated activation of stress kinases such as 408 JNK or IkK under immune or metabolic stimuli thus linking lipid signalling to pro-409 inflammatory signalling and anti-insulin action [START_REF] Hotamisligil | Uncoupling of obesity 499 from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid 500 binding protein[END_REF]. It has been 410

hypothesised that the presence of FABP4 in periods of feast and famine may have 411 been beneficial to maintain a strong immune response and adipose tissue in fitting 412 with the concept of 'thrifty' phenotype to survive [START_REF] Auwerx | PPAR-gamma: a thrifty transcription 447 factor[END_REF].

413

However, with increasing prevalence of excessive caloric intake, decreased energy 414 expenditure and high stress lifestyle, FABPs may not be able to maintain 415 inflammatory or metabolic homeostasis and the presence of these proteins may 416 actually aid in the formation of obesity, dyslipidemia and inflammatory responses 417 [START_REF] Hotamisligil | Uncoupling of obesity 499 from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid 500 binding protein[END_REF]. Combined with the prevalent increased pro-inflammatory 418 IL-6 production in elderly individuals, these alterations in inflammatory homeostasis 419 have serious and detrimental implications for health in ageing populations.

  has demonstrated that polymorphisms in the MT coding region are 56 involved in regulating zinc release by MT and are associated with longevity (Cipriano 57 et al. 2006). Cipriano et al. have shown that carriers of the C-allele at position +647 58 of the MT1a gene show enhanced zinc release, low metallothionein levels, and 59 reduced IL-6 plasma concentrations. In addition, metallothionein gene polymorphisms 60 have been associated with cardiovascular disease, hyperglycaemia and mild zinc 61

  acetoxymethylated (AcOM-DEA/NO, Calbiochem, VWR 125 International s.r.l., Milan, It) normalized to the minimum fluorescence. 126 127

130

  Samples probed with an irrelevant (isotypic) antibody (clone MOPC21, IgG1k, 131 Sigma-Aldrich, MI, Italia) in conjunction with the fluorescein-conjugated secondary 132 antibody served as staining controls. The MT-specific fluorescence of the un-gated 133 population was determined by subtracting the mean fluorescence intensity obtained 134 with the isotypic antibody from the one obtained with the MT-specific antibody. nucleotide polymorphism (SNPs), IL-6 -174 G/C (rs1800795) and MT1a 138 +647 A/C (rs11640851) were screened. The MT1a SNP corresponded to an amino 139 acid transition from asparagine in the MT1a-A (adenine) to threonine in the MT1a-C 140 (cytosine). PCR-restriction fragment length analysis (PCR-RFLP) for this SNP was 141 performed using the following sense 5'-ACTTGGCTCAGCCCCAGATT-3' and 142 antisense 5'-CACTCAGCTGGCAGCAT-TTG-3' primers.

9

  The human oligonucleotide library was purchased from Sigma-Compugen (Oligo 175 library, release 1.0, Jamesburg, NJ, USA) with a further additional set of 700 176 oligonucleotides custom synthesised according to the manufacturer's standard design 177 guidelines. Oligonucleotides were printed onto Codelink slides (GE Healthcare, 178

  gain an understanding of which functional cellular processes may be most 234 affected by exogenous zinc treatment, the 61 genes that were significantly altered in 235 the presence of zinc were visualised by Ingenuity Pathway Analysis TM . Ingenuity 236 entry tool systematically encodes findings presented in peer-reviewed scientific 237 publications into ontologies, or groups of genes /proteins related by common function.238 A molecular network of direct physical, transcriptional, and enzymatic interactions 239 was computed from this knowledge base. The resulting network contains molecular 240 relationships with a high degree of connectivity and every gene in the network is 241 supported by published information. Genes identified through microarray analysis 242 (focus genes), network score (probability of network being assembled by chance 243 alone), and main cellular functions of the network are listed in Figure 1a. The two A C C E P T E D M A N U S C R I P T 12 statistically significant molecular networks assembled using Ingenuity Pathway 245 Analysis (IPA) profiling of genes differentially expressed in the presence of zinc, 246 including interacting partners, are depicted in Figure 1b and Figure 1c, respectively. 247 Genes demonstrated to be up-regulated or down-regulated in the presence of zinc are 248 coloured in red and green, respectively. Class of function for each of the genes, 249 including enzymes, growth factors, and receptors, are depicted by nodal shape, as 250 identified in the figure legend. Interestingly, IPA identified interactions between IL-6 251 (gene expression decreased by zinc treatment) and metalloproteases (MMP9, 252 MMP19) and pro-inflammatory mediators (IL1R2, CXCL3), all of which were down-253 regulated following zinc treatment. IPA further identified zinc-dependent down-254 regulation of several obesity-related genes: leptin, NR1H3 (LXR-α) and fatty acid 255 binding protein 3 (FABP3). These data suggest that zinc may act as an important 256 anti-inflammatory and anti-obesity agent. These results are consistent with the known 257 age-dependent increases in pro-inflammatory status and fat mass.

  test the hypothesis that variability in IL-6 -174 and MT1a +647 loci 260 influences the effects of zinc on the genomic level, donors were stratified by IL-6 and 261 MT1a genotype. Individuals were determined to be either one of three genotypes: (1) 262 MT1a +647 C-allele-/IL-6 -174-C-allele-positive carriers (C+C+), (2) MT1a +647 C-263 allele-positive/IL-6 -174-C-allele negative carriers (C+C-) or C-allele negative in both 264 MT1a +647 and IL-6 -174 loci (C-C-). These three genotypes were distributed evenly 265 throughout the donor population analysed, as shown in

  -C-individuals, suggesting this pathway was regulated in a zinc-295 dependent IL-6/MT-independent manner. The interferon signalling pathway was 296 regulated by zinc in only C-C-individuals, suggesting this pathway be co-ordinately 297 regulated by MT and IL-6 interactions.

  assess whether variability in the IL-6 and MT1a loci influences zinc 300 homeostasis or response to zinc supplementation, cellular zinc ion availability, NO-301 induced release, and metallothionein expression were determined for each genotype 302 before and after zinc treatment, as previously described[START_REF] Malavolta | Single and three-color flow 520 cytometry assay for intracellualr zinc ion availability in human lymphocytes with 521 Zinpyr-1 and double immunofluorescence: relationship with metallothioneins[END_REF]. Zinc 303 availability (shown as mean normalized fluorescence) in lymphocytes (Figure 4a) 304 and monocytes (Figure 4c) and NO-induced zinc release (shown as difference in the 305 mean normalized fluorescence after the addition of AcOM-DEA/NO) from 306 lymphocytes (Figure 4b) and monocytes (Figure 4d), was determined before and 307 after in vitro culture with exogenous zinc, for MT1a/IL-6 C+C+, C+C-, and C-C-308 genotypes. The average total metallothionein protein expression across donors for 309 each genotype as determined by mean fluorescence intensity is shown in Figure 4e. 310 While increases were observed in zinc availability in lymphocytes and monocytes, 311 and in metallothionein expression following zinc treatment, no statistically significant 312 difference was observed between the IL-6 and MT1a genotypes. Similarly, there was 313 no significant difference between IL-6 and MT1a C+ and C-carriers in zinc 314 availability, NO-induced release of zinc, or metallothionein expression at the basal 315 level, prior to zinc treatment. We observed no significant genotype-dependent 316 difference between basal-and zinc-stimulated levels of zinc availability, NO-induced 317 zinc release or metallothionein expression. Additionally, in the case of NO-induced 318 zinc release in monocytes and lymphocytes derived from C+C+ carriers, we did not A C C E P T E D M A N U S C R I P T 15 observe a significant increase in either parameter following zinc treatment. This was 320 due to a surprising reduction of these parameters in some experimental samples and 321 concomitant increased variability within this group. Lack of increased NO-stimulated 322 zinc release in C+C+ carriers following zinc treatment corresponded with slightly 323 increased basal levels of NO-stimulated zinc release in C+C+ monocytes compared to 324 IL-6 -174 C-carriers, although this was not significant. These data suggest that the 325 most significant responses to zinc treatment on the level of NO-stimulated zinc 326 release in monocytes and lymphocytes occur in IL-6 C-carriers, which is consistent 327 with the increased zinc-dependent gene expression in this group.

332

  loci influenced zinc-regulated gene expression in ageing. The hypothesis was taken 333 that an anti-inflammatory molecule such as zinc might impact positively on IL-6 334 production in the elderly. Since MT1a polymorphisms are associated with increased 335 IL-6 production and could potentially impact on zinc homeostasis, we further 336 segmented the elderly population recruited for investigation based on MT1a 337 polymorphism. It was thought that MT1a genotype may exacerbate the IL-6 338 phenotype. By combining these two genetic polymorphisms we were able to identify 339 an elderly sub-population who responded significantly to zinc treatment through 340 modulation of inflammatory and metabolic gene expression profiles. This approach 341 offers substantial benefit over attempting zinc intervention in a non-segmented 342 population as many of the general ageing population are zinc sufficient. It is hoped 343 that using nutrigenomic or nutrigenetic approaches to identify sub-populations who 344

  that were differentially regulated on the basis of IL-6 genotype, the most 349 interesting belong to either the IL-6 signalling pathway itself or are involved in 350 regulation of energy balance/obesity, including IL-6, IL-1R2, IL-1, leptin, and 351 FABP4. These data are consistent with the established links between serum IL-6 352 levels, IL-6 promoter polymorphisms and obesity (Klipstein-Grobusch et al. 2006; Qi 353 et al. 2007; Wernstedt et al. 2004). Zinc supplementation has previously been shown 354 to improve glycemic control in db/db and ob/ob mice (Begin-Heick et al. 1985, Simon A C C E P T E D M A N U S C R I P T 17 and Taylor 2000). In fact, zinc-deficient mice have multiple diabetogenic traits 356 including impaired glucose tolerance, insulin resistance and impaired insulin 357 signalling (Cunnane et al. 1984, Gomot et al. 1992). A study carried out in obese 358 children associated lower zinc serum concentrations with abdominal obesity (Beattie 359 et al. 1998, Marreiro et al. 2004). Conversely, zinc supplementation has been 360 demonstrated to increase insulin secretion and to improve glucose control in db/db 361 and ob/ob mice (Begin-Heick et al. 1985, Simon and Taylor 2000).

369

  animals is characterised by elevated circulating leptin, a hormone that is produced by 370 adipocytes[START_REF] Rebuffe-Scrive | Regional fat distribution 549 and metabolism in a new mouse model (C57Bl/6J) of non-insulin dependent 550 diabetes mellitus[END_REF][START_REF] Van Heek | Diet-induced obese mice 557 develop peripheral, but not central, resistance to leptin[END_REF]). Here we show that 371 the addition of exogenous zinc led to increased MT expression concomitant with 372 decreased leptin gene expression in IL-6 C-allele negative individuals. Wernstedt et 373 al. have previously demonstrated that the IL-6 -174 C allele was associated with 374 higher BMI and higher serum leptin levels (Wernstedt et al. 2004). Since several 375 adipose-derived cytokines including leptin, tumor necrosis factor-α, and IL-6 are up-376 regulated in obesity and contribute to the development of diabetes and vascular 377 disease, the findings that zinc could potentially decrease expression of these 378 molecules is important for obesity-related conditions. Taken together, these data 379 suggest that zinc intervention may be useful to restore normal levels of leptin and IL-6 A C C E P T E D M A N U S C R I P T 18 and could potentially impact positively on adipose mass in individuals with prostudy, we demonstrated that zinc treatment resulted in decreased 384 FABP4 gene expression. Differential regulation of FABP4 has previously been 385

  396 obesity, these mice maintained euglycemic control and demonstrated improved 397 peripheral insulin sensitivity. These data indicate that reductions in leptin and FABP4 398 by dietary zinc in IL-6 C-allele negative individuals may protect against insulin 399 resistance and suggest an important mechanism by which zinc may protect against 400 development of type 2 diabetes.

  its role in the regulation of energy balance and obesity, evidence 403 obtained recently indicates that FABPs may serve as master regulators in the control 404 of cellular inflammatory responses through regulation of JNK and PPARγ (Hirosumin A C C E P T E D M A N U S C R I P T 19 et al

Figure 1 :

 1 Figure Legends

  Figure 2: Genes differentially expressed in MT1aC+/IL-6 C+, MT1aC+/IL-6 C-,

ion availability in lymphocytes and monocytes 117

	105
	106	mononuclear cells (PBMC) were recovered. Cell cultures were prepared in zinc-free
	107	RPMI medium containing 10% of heat-inactivated FCS, 4 mM L-glutamine, 100
		units/ml penicilin and streptomycin (Invitrogen, San Giuliano Milanese, MI, Italia).

108

Cells were incubated for 24 h (37C, 5% CO 2 ) with addition of zinc 50 M (zinc-109 enriched medium). At the end of the 24 h of culture, cells were recovered, washed 110 three times with RPMI medium, and counted. An aliquot of 2 x 10 6 cells was used for 111 the determination of intracellular available zinc and MT expression by flow 112 cytometry. Another aliquot (3-4 x 10 6 cells) was frozen and preserved for microarray 113 analysis. The research protocol was approved by the Ethical Committee of INRCA 114 (Ancona, Italy).

115 116 Zinc

Table 1 .

 1 In order to examine the effect of zinc supplementation on gene expression profiling in Gene expression profiling of cells cultured in the 225 presence of zinc was compared to cells cultured in the absence of exogenous zinc.

	202
	203	expressed following zinc treatment. Ingenuity TM Pathway Analysis 3.0 was utilised to
	204	assemble functional networks altered by zinc status (Ingenuity TM Systems, CA, USA).
	205
	206	Real-time RT-PCR
		Taqman (Applied Biosystems, Applera, UK) probes were used for RT-PCR (IL6:

180

labelling system (Hatfield, PA, USA) according to the manufacturer's standard 181 protocol. Reverse transcription was performed using Powerscript RT (BD 182 Biosciences, Oxford, UK) with a final dNTP concentration of 0.5mM each and using 183 the 5pMol/ul modified oligo dT primer provided (Genisphere, Hatfield, PA, USA).

184

Hybridisation was performed using a Lucidea SlidePro (GE Healthcare, Amersham, 185 UK). Dendrimer hybridisation was performed with dextran concentration reduced to 186 2.5% final (w/v), hybridisation time was reduced to 4hrs at 50 o C. Scanning was 187 performed using a Perkin Elmer Scanarray 4000XL scanner (Buckinghamshire, UK) 188 for both Cy3 and Cy5. Image analysis and data extraction was performed using 189 Bluefuse V3.2 (Bluegnome Ltd, Cambridge, UK). Manual exclusion was further 190 applied to remove outliers due to contamination. 191 192 Bioinformatics Analysis 193 Data sets were analysed using GeneSpring v7.2 (Silicon Genetics, CA, USA). Loess 194 normalisations were applied to all data sets. A subset of genes for data interrogation 195 was generated that excluded controls, spots of poor quality, and gene probes that were 196 present in less than 50% of samples. From these selected genes, relative expression 197 compared to untreated cells was determined and genes differentially expressed by a 198 minimum of two-fold in the presence of zinc were determined. One-way, parametric, A C C E P T E D M A N U S C R I P T 10 ANOVA tests were performed followed by Benjamini and Hochberg multiple test 200 correction with a false discovery rate of 0.05. Microsoft Excel templates were 201 prepared for each of the genotypes containing genes that were over-and under-207 Hs00174131_m1; FABP-4: Hs00609791_m1; leptin: Hs00174877_m1; IL-1β: 208 Hs00174097_m1; IL-1R2: Hs00174759_m1; GAPDH: Hs99999905_m1). RNA was 209 prepared as before and in vitro transcription was performed using 1µg total RNA and 210 random hexamer primers (Invitrogen, Paisley, UK). The Bio-rad I-Cycler (Bio-rad, 211 Hercules, CA) with FAM-490 system detection was used for real time RT-PCR. PCR 212 thermocycler conditions were 50°C for two minutes, 90°C for two minutes followed 213 by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Samples were run in 214 triplicate with test probes and the control gene human GAPDH to control for 215 differences in amount of starting material. Fold-changes (compared to untreated 216 cells) were calculated by normalising the test crossing threshold (Ct) with the 217 GAPDH amplified control Ct. 227 presence of zinc. Of these, 17 were up-regulated and 44 down-regulated. To confirm 228 that these genes identified were truly differentially expressed, stringent data analysis 229 was performed. After exclusion of genes with missing spots, spots of poor quality, 230 genes below detection threshold, and ANOVA testing, these 61 genes were 231 considered to be truly differentially expressed.

Table 1 .

 1 The MT1a +647 C-

	266
	allele-negative/IL-6 -174-C allele positive carriers (C-C+) were not considered for

267

analysis due to high variability in gene expression in this group (data not shown for 268 n=6 individuals) which we were not able to address by recruiting more individuals

Proc. Natl. Acad. Sci. U S A. 102, 6843-8.

Acknowledgements 431 432

We thank Ann Scarborough for technical assistance. This study was supported by