Nikola Serafimovski
email: n.serafimovski@ed.ac.uk

Sinan Sinanović
email: s.sinanovic@ed.ac.uk

Marco Di Renzo
email: marco.direnzo@lss.supelec.fr

Harald Haas
email: h.haas@ed.ac.uk

Dual-hop Spatial Modulation (Dh-SM)

In this paper, we introduce Dual-hop Spatial Modulation (Dh-SM). We look at the effect that Dh-SM has on the required signal to noise ratio (SNR) at the destination and how it can help alleviate the multi-hop burden in the system. Initial biterror-ratio (BER) results comparing the performance of Dh-SM with orthogonal decode-and-forward (DF) are presented where Dh-SM is shown to have up to a 10 dB SNR advantage.

I. INTRODUCTION

Spatial modulation (SM) is a recently proposed approach to multiple-input-multiple-output (MIMO) systems which entirely avoids inter-channel interference (ICI) and requires no synchronisation between the transmit antennas, while achieving a spatial multiplexing gain [START_REF] Mesleh | Interchannel Interference Avoidance in MIMO Transmission by Exploiting Spatial Information[END_REF]. This is performed by mapping a block of information bits into a constellation point in the signal and spatial domains [START_REF] Mesleh | Spatial modulation[END_REF]. In SM, the number of information bits, k, that are encoded in the spatial domain is directly related to the number of transmit antennas N t ; in particular N t = 2 k . This means that the number of transmit antennas must be a power of two unless fractional bit encoding is used [START_REF] Serafimovski | Fractional Bit Encoded Spatial Modulation (FBE-SM)[END_REF]. It should also be noted that SM is shown to outperform other MIMO schemes in terms of bit-error-ratio (BER) [START_REF] Mesleh | Spatial modulation[END_REF]. The presented work proposes the use of SM as a possible relaying technique.

The basic relaying problem can be reduced to the simple inability of a single transmitter to reach its intended target with the necessary signal to noise ratio (SNR). There are several approaches to this problem. On one hand, the orthogonal amplify-and-forward (AF) utilises the relay antenna as a simple amplifier. Any signal received by the relay at time instance t 1 is amplified and retransmitted at instance t 2 forming a non-regenerative system. On the other hand, the orthogonal decode-and-forward (DF) algorithm decodes the received signal at the relay, then re-encodes and retransmits this information establishing a regenerative system. Outage probabilities, mutual information calculations and transmit diversity bounds for AF and DF relaying are derived in [START_REF] Laneman | Cooperative Diversity in Wireless Networks: Efficient Protocols and Outage Behavior[END_REF] with end to end performance being considered in [START_REF] Hasna | End-to-end performance of transmission systems with relays over rayleigh-fading channels[END_REF]. Taking into consideration the above relaying protocols, the use of SM is proposed to provide additional power and capacity gains over the non-cooperative AF and DF systems. Let us assume a basic system as shown in Fig. 1, where transmissions are carried out at 2 bits/s/Hz in the signal domain and only a single transmit antenna is active at the source. In orthogonal AF and DF two time slots are needed for the relevant information to reach the destination node, effectively halving the sourcedestination spectral efficiency to 1 bit/s/Hz. Dual-hop Spatial Modulation (Dh-SM) can partially mitigate this effect. While maintaining a fixed signal constellation, Dh-SM can utilise the spatial domain to transmit additional information bits. Since the receiver decodes the channel used for the transmission, it can determine the transmitting antenna and, in so doing, decode the bits used to activate the particular antenna. This serves to increase the source to destination spectral efficiency i.e. almost halving the multihop burden as will be explained in Sectin II. Alternatively, since some of the data in SM is transmitted in the spatial domain, a lower order modulation scheme can be used for signal domain transmission which in turn leads to a lower transmit power requirment. This is a unique advantage that Dh-SM has when compared to all other relaying systems and results in a decreased bit error ratio at the destination for the same transmit power i.e. Dh-SM increases the coding gain of the system.

In [START_REF] Jeganathan | Spatial Modulation: Optimal Detection and Performance Analysis[END_REF][START_REF] Handte | BER analysis and optimization of generalized spatial modulation in correlated fading channels[END_REF][START_REF] Younis | Generalized Spatial Modulation[END_REF], analytical bounds for the BER performance of SM are derived. Each work considers the channel and the signal symbol as a joint input variable and averages across the channel to achieve a closed form solution. In this work, besides introducing Dh-SM, we validate the results of this work with the union bound based approach originally presented in [9, Eq. ( 8)].

In the remainder of the paper we introduce the system model in Section II, provide the theoretical framework in Section III, show and discuss the numerical results in Section IV and conclude the paper in Section V.

II. SYSTEM MODEL

In the following work we assume a three node scenario as shown in Fig. 1. While AF, DF and Dh-SM utilise a single transmit antenna at any instance, Dh-SM requires that the transmitter has more than one transmit antennas. Since in this work we seek to characterise the behaviour of SM in a dual-hop scenario, we compare its performance in terms of BER to non-cooperative DF. The source broadcasts a signal constellation symbol, x. The received signal is given by: y j = h ij x + η, where j is the index of the receive and i is the index of the transmit antenna, h ij is the channel coefficient of the link between the active antenna i and the receiving is the expectation with respect to the set of signal constellation points. The estimated symbol at the relay in the DF system using maximum-ratio-combining (MRC) is given by:

x ℓ est = h i kℓ † ȳkℓ h i kℓ † h i kℓ (1)
(•) † denotes the complex conjugate and h i kℓ = h i1 . . . h iN ℓ r is a vector composed of the single tap channel coefficients from antenna i on the transmitting entity k to the receiving entity ℓ with N ℓ r number of receive antennas. The transmitting entity is either the source, s, or the relay, r, while the receiving entity is the relay or destination, d i.e. k ∈ {s, r} and ℓ ∈ {r, d}. The vector ȳkℓ is comprised of the symbols at each of the receive antennas at node ℓ. Finally, x ℓ est is passed through a maximum likelihood (ML) detector to obtain the original bit sequence.

The basic idea of SM is to map blocks of information bits into two information carrying units [START_REF] Mesleh | Spatial modulation[END_REF]: i) a symbol, chosen from a complex signal-constellation diagram, and ii) a unique transmit-antenna index, chosen from the set of transmitantennas in the antenna-array i.e. the spatial-constellation diagram. The working principle of SM is exemplified in Fig. 2.

Throughout this paper, we consider a ML decoder, which computes the Euclidean distance between the received signal ȳkℓ and the set of all possible received signals, selecting the closest one [START_REF] Jeganathan | Spatial Modulation: Optimal Detection and Performance Analysis[END_REF]:

(x est , n k t ) = argmin ȳkℓ -x j h i kℓ 2 F ∀x j ∈ X ∀i ∈ {1 . . . N k t } where the pair (x est , n k t )
is formed from the estimated symbol x est emitted from antenna n k t at node k, x j is the current symbol being evaluated from the set of possible constellation points X , N k t is the number of available transmit antennas on node k and || • || F is the Frobenius norm.

A. Example

Let us again consider the system presented in Fig. 1, but now with a single source to relay transmit antenna and four relay to destination transmit antennas. In this case 2 bits can be sent in the spatial domain and 2 more in the signal domain on the relay to destination link. The use of SM on the relay to Fig. 2. If we wish to transmit four bits, the first two bits define the spatial-constellation point which identifies the active antenna, while the remaining two bits determine the signal-constellation point that is to be transmitted.

destination link enables the system to operate at 4 bits/s/Hz on that link. Since the system remains unchanged in the source to relay link, the source can transmit to the relay in the first two time slots a total of 4 bits. The relay can then transmit those 4 bits to the destination in the third slot by making use of the spatial domain. The use of Dh-SM results in 4 bits going from the source to the destination in three time slots and an end-to-end average spectral efficiency of 1.33 bits/s/Hz; a 33% improvement over standard AF and DF.

Alternatively, Dh-SM can be used to improve the bit-errorratio of the system by transmitting a lower order modulation signal-symbol. We quantify the coding gains in Section IV-B.

III. ANALYTICAL MODELLING

The scenario presented in Fig. 1 represents a well known orthogonal relaying system. One major distinction is the use of multiple transmit and receive antennas at the relay. The end-toend performance of a two-hop wireless communication system with non-regenerative (AF) and regenerative (DF) relays over a Rayleigh-fading channel is presented in [START_REF] Hasna | End-to-end performance of transmission systems with relays over rayleigh-fading channels[END_REF]. The authors develop a closed form expression for the average BER of AF given in terms of the system's moment generating function (MGF). The system performance in terms of outage probability and BER demonstrate that DF systems perform better than AF at both low and high average SNRs in a Rayleigh fading environment. For this reason, we limit our comparison and only look at Dh-SM's performance relative to DF. In this work we aim to show that the use of spatial modulation in a fixed relay system provides a significant coding gain.

Starting from the system model presented in Section II, for the case of DF, the bit error probability averaged over the independent channel SNRs can readily be expressed as shown in [START_REF] Mesleh | Spatial modulation[END_REF], since the signal undergoes two stages of decoding [START_REF] Gagliardi | Introduction to Communication Engineering[END_REF],

P b (E sd ) = P b (E sr ) + P b (E rd ) -2P b (E sr )P b (E rd ). (2) P b (•)
is the average BER with average energy E kℓ between nodes k and ℓ. In the decode and forward case, the overall bit error ratio for a dual-hop system is a function of the individual links, meaning that if a system performs better in terms of BER on the individual links, it will also perform better for the dualhop when we consider that both P b (E sr ) and P b (E rd ) must be less than 1/2. To show this we look at the directional derivative of P b (E sd ) with respect to P b (E sr ) and P b (E rd ). We can define a unit vector -→ u = < α, β > where α and β are non-negative coefficients defining the direction of the derivative.

∇-→ u P b (E sd ) = α (1 -2P b (E rd )) + β (1 -2P b (E sr )) . (3)
Looking at [START_REF] Serafimovski | Fractional Bit Encoded Spatial Modulation (FBE-SM)[END_REF] we see that the function is monotonically increasing with respect to the individual error probabilities since {P b (E sr ), P b (E rd )} ∈ [0, 1/2]. We now look at the expressions for these error probabilities. It should be noted that since the overall system error depends solely on the error of the individual links, we proceed to analyse the error expressions for the arbitrary k to ℓ link.

The BER of an M quadrature amplitude modulation (QAM) across multiple fading channels is given in [START_REF] Laneman | Cooperative Diversity in Wireless Networks: Efficient Protocols and Outage Behavior[END_REF] where M is the size of the QAM constellation and N ℓ r depends on which link is considered. The generalized expression for the average BER of a single link between nodes k and ℓ using QAM modulation and Gray coding is given by (4) [START_REF] Simon | Digital Communication over Fading Channels[END_REF].

P b (E kℓ ) ∼ = A √ M/2 b 1 π π/2 0 M γkℓ c 2 sin 2 (θ) N ℓ r dθ (4) A = 4 √ M -1 √ M log 2 (M ) c = - 3 log 2 (M )(2b -1) 2 M -1 M γ (s)
is the moment generating function of the fading channel with γ being the average SNR at the receiver. The moment generating functions for different channel fading models can be found in [START_REF] Simon | Digital Communication over Fading Channels[END_REF]. In particular, we look at a Rayleigh fading channel and M γ (s) is given by:

M γ (s) = 1 1 + sγ
Similarly, because Dh-SM is in principle a DF system, its BER can be represented by [START_REF] Mesleh | Spatial modulation[END_REF], where the individual error probabilities are those of individual SM links.

The BER of SM using the optimal detector can be bounded using union bound methods and is given by ( 5):

P b (E kℓ ) ≤ E H   x,nt PEP (x, n t , x, nt )   (5)
where we define PEP (x, n t , x, nt ) to be the pairwise error probability between the symbol x emitted from antenna n t being detected as symbol x emitted by antenna nt . E H [•] represents the expectation of the system with respect to the channel. Given this formulation, the symbol based union bound for (5) can be expressed as [START_REF] Jeganathan | Spatial Modulation: Optimal Detection and Performance Analysis[END_REF], where work in [START_REF] Di Renzo | A General Framework for Performance Analysis of Space Shift Keying (SSK) Modulation for MISO Correlated Nakagami-m Fading Channels[END_REF] shows the tightness of this approach.

P b (E kℓ ) ≤ M x=1 M x=1 N k t nt=1 N k t nt=1 PEP kℓ (x, n t , x, nt ) 2 M N k t -1 . (6)
In [9, Eq. ( 8)] the pairwise error probability conditioned on the channel between the two communicating nodes is given as:

PEP kℓ (x, n t , x, nt ) = Q     h nt kℓ x -h nt kℓ x 2 2σ 2     (7)
where the symbol x is transmitted from antenna n t . Given this analytical modelling, we proceed to analyse the performance of Dh-SM and DF in terms of their BER.

IV. NUMERICAL ANALYSIS

The aim of this section is to compare the performance of Dh-SM with conventional relaying utilising M-QAM under a variety of conditions. In particular, the presented results depict the different behaviour of the system when:

• the number of transmit antennas is changed at the source,

• the number of transmit antennas is changed at the relay,

• the number of receive antennas is changed at the relay,

• the number of receive antennas is changed at the destination and, • under non-symetric channel gain conditions.

A. Simulation Setup

A frequency-flat Rayleigh fading channel with no correlation between the transmitting antennas and additive white Gaussian noise is assumed. MRC in combination with ML detection is used in the DF system, with the ML detector in [START_REF] Jeganathan | Spatial Modulation: Optimal Detection and Performance Analysis[END_REF] being used for Dh-SM. Perfect channel state information (CSI) is assumed at the receiving node, with no CSI at the transmitter. Only one of the available transmit antennas at the source and relay nodes of Dh-SM is active at any transmitting instance. Since part of the data is encoded in the spatial domain, Dh-SM uses a lower order modulation symbol but the energy per symbol is equivalent to that in the DF system.

B. Results

In the legend on each figure ,An(k,ℓ) represents the analytical BER on the link between nodes k and ℓ, while Sim(k, ℓ) is the simulation result. To describe the behaviour of DF we use (4), whereas (6) bounds the behaviour of Dh-SM. Throughout the discussion we refer to the coding gain as the the difference between the SNR levels of the two systems required to reach the same BER. We begin by looking at the effects of additional antennas at the transmitter. In particular, Fig. 3 shows that when the source to relay and relay to destination channel conditions are comparable, Dh-SM exhibits over a 2 dB coding gain when compared to DF. It should be noted that as any DF system, Dh-SM is susceptible to bottlenecks. In this case, the two transmit antennas at the relay and the two receive antennas at the relay and destination limit the system's performance. The effect of the number of receive antennas is quantified below. Indeed the addition of two more antennas at the source increases the advantage of Dh-SM only by about 0.4 dB and, more noticeably, the addition of 28 more transmit antennas results in a mere 0.6 dB gain. Similarly, looking at Fig. 4, we can see that if we remove one of the bottlenecks i.e. operate with 32 transmit antennas at the source, the coding gain of 3.5 dB achieved with only 2 transmit antennas at the relay is marginally increased by about 0.5 dB with 4 transmit antennas.

A further 1 dB coding gain is achieved by using 32 transmit antennas at the relay. It must be noted that systems with more available antennas still exhibit better performance in terms of BER, although the coding gains achieved with every additional antenna are diminishing. We now investigate the behaviour of Dh-SM with respect to the number of receive antennas. As Fig. 5 shows, Dh-SM performs better as more receive antennas are added to the system, irrespective of which node they are added to. Despite the minor coding gains observed, the system is still limited by its worse performing hop in terms of the BER since the diversity of the overall system is min{N r r , N d r }. Looking at Fig. 5, we can see that Dh-SM has a 2 dB gain with respect DF when there are 2 receive antennas at both the relay and the destination. When there are 2 and 4 receive antennas at the relay and destination nodes respectively, Dh-SM has a 2.5 dB gain with respect to DF, i.e. the performance of Dh-SM improves by 0.5 dB with the addition of 2 receive antennas at the destination. Similarly, when there are 4 antennas at each of the nodes, Dh-SM gains an additional 0.5 dB which results in a 3 dB better performance compared to DF. Similar to the effects observed with the number of transmit antennas, systems with more receive antennas still exhibit better performance in terms of BER, although the coding gains achieved with every additional antenna are diminishing. It should be noted that all coding gains observed in the Dh-SM system are in addition to the diversity gains resulting from the increase in the number of receive antennas experienced by both systems.

With the effect of the number of antennas analysed, Fig. 6 shows the effects of the non-symmetric channel gains, i.e. γsr = γrd . Since Dh-SM relies on sequential detection, from Fig. 6 we can see that both systems exhibit better performance when the average received SNR on the source to relay link is greater than the average received SNR on the relay to the destination link. As can be seen, however, the difference in the average received SNR merely causes a shift in the performance of the entire system. This means that the effects observed in the above discussion extend to arbitrary channel gain conditions and system geometry, given that on the channel vectors, h i kℓ , from transmitting node k to receiving node ℓ are sufficiantly distinct from each other.

With the individual effects of the number of transmit and receive antennas analysed, Fig. 7 shows that Dh-SM can exhibit between 5 and 10 dB gains compared to DF when the number of receive antennas at both the relay and the destination is increased to 4 and N s t = N s t . These gains are larger than those presented in Fig. 3 and Fig. 4, due to the greater number of receive antennas at both the relay and destination, which provide a significant increase in spatial diversity for both systems and additional coding gains for Dh-SM i.e. all bottlenecks have been removed from the system.

V. CONCLUSION

In this work the application of SM in a dual-hop, noncooperative scenario is considered. In Dh-SM the spatial domain is utilised to transmit extra information bits which help alleviate the multihop burden. Dh-SM is also shown to provide coding gains by lowering the number of bits sent in the signal domain. The union bound method is used to bound the BER behaviour of SM and provide a good estimate for the potential performance of Dh-SM.

It is demonstrated that the application of SM in a relaying scenario results in better end-to-end system performance when compared to non-cooperative DF. The coding gain is increased as the number of transmit antennas is increased at either the source or relay nodes by about 2 dB. Furthermore, the coding gain is also increased with the number of receive antennas added at either the relay or destination nodes resulting in gains of around 3 dB when compared to DF. From this, it can be seen that Dh-SM has the potential to provide substantial spectral efficiency and coding gains in future wireless relay networks.

Fig. 1 .

 1 Fig. 1. Dual-hop spatial modulation

Fig. 3 . 2 DF 2 Fig. 4 .

 3224 Fig.3. Average bit error ratio when γsr = γrd . The average source to destination spectral efficiency is equal to 3.5 bits/s/Hz. The arrow on the figure indicates the progression of the curves going from N s t = 32 as the leftmost dashed curve to N s t = 2 as the rightmost dashed curve.

Fig. 5 .

 5 Fig.5. Average bit error ratio when γsr = γrd . The average source to destination spectral efficiency is equal to 3.5 bits/s/Hz.

Fig. 6 .Fig. 7 .

 67 Fig.6. The average source to destination spectral efficiency is equal to 3.5 bits/s/Hz.

ACKNOWLEDGEMENT We gratefully acknowledge support from the Engineering and Physical Sciences Research Council (EP/G011788/1) in the United Kingdom for this work. Professor Harald Haas acknowledges the Scottish Funding Council support of his position within the Edinburgh Research Partnership in Engineering and Mathematic between the University of Edinburgh and Heriot Watt University.


Naseem Khan

Bernard Rawlings

Patrick Caffrey
email: patrick.caffrey@ucd.ie

A labile point in mutant amphotericin polyketide synthases

Keywords: amphotericin B, polyketide synthase, ketoreductase, polyenyl-pyrone

Streptomyces nodosus produces the antifungal polyene amphotericin B.

Numerous modifications of the amphotericin polyketide synthase have yielded new analogues. However, previous inactivation of the ketoreductase in module 10 resulted in biosynthesis of truncated polyketides. Here we show that modules downstream of this domain remain intact. Therefore, loss of ketoreductase-10 activity is sufficient to cause early chain termination. This modification creates a labile point in cycle 11 of the polyketide biosynthetic pathway.

Non-extendable intermediates are released to accumulate as polyenyl-pyrones.

Introduction

Amphotericin B 1 (Fig. 1) is used in clinical medicine for treatment of serious systemic fungal infections and visceral leishmaniases. It is obtained from fermentation cultures of Streptomyces nodosus. Both chemical modification and engineered biosynthesis have generated analogues with reduced toxicity [START_REF] Caffrey | Biosynthetic engineering of polyene macrolides for generation of improved antifungal and antiparasitic agents[END_REF]. The aglycone 19-deoxy-19-oxo-amphoteronolide B 2 (Fig. 1) has potential as a starting material for chemical synthesis of derivatives with altered sugar residues. Small quantities of this compound have been obtained by chemical deglycosylation of amphotericin B [START_REF] Kennedy | Degradation of amphotericin B: Cleavage of the glycosidic linkage with aluminum amalgam [Al(Hg)] of dichlorodicyanoquinone (DDQ)[END_REF]. Biosynthesis of 19-deoxy-19-oxo-analogues was expected to result from inactivation of ketoreductase (KR) domain 10 of the amphotericin polyketide synthase (PKS) [START_REF] Caffrey | Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes[END_REF]. However, we recently found that KR10 mutants of S. nodosus produce truncated polyketide intermediates instead of full-length macrolactones (Fig. 1) [START_REF] Murphy | Isolation and characterisation of amphotericin B analogues and truncated polyketide intermediates produced by genetic engineering of Streptomyces nodosus[END_REF]. The KR10-1 strain synthesised the pentaenyl-pyrone 3 as a single product whereas the KR10-2 strain gave both the heptaenyl-pyrone 4 and the tetraenyl-pyrone 5 (Fig. 1). These new compounds were produced in good yields of up to 20 mg purified material per litre of culture.

Preliminary PCR analysis of strain KR10-1 suggested that modules 5 and 6 had been precisely deleted from the AmphC protein that synthesises most of the polyene unit. This unexpected recombination event was not related to targeted replacement of the KR10 coding sequence, which is located at an approximate distance of 80 kb along the chromosome. The loss of these two modules accounts for the shorter chain length of pentaenyl-pyrone 3. Module 5 contains a partly functional enoylreductase domain [START_REF] Caffrey | Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes[END_REF] that causes formation of both heptaene 4 and tetraene 5 by strain KR10-2.

From the structures of the new pyrone compounds, it can be deduced that no ketoreduction occurs during cycle 10, as expected for a KR10 mutant (Fig. 2). The unprocessed -ketoacyl chains migrate from ACP10 to KS11 and incorporation of methylmalonyl CoA-derived extender units gives polyenyl-2-methyl-3, 5-diketoacyl-ACP11 thioester intermediates (Fig. 2). These products undergo no further enzymatic processing, and are released to accumulate as pyrones. This finding was unexpected because inactivation of KR12 or KR16 did not block subsequent steps in polyketide chain assembly [START_REF] Power | Engineered synthesis of 7-oxo-and 15-deoxy-15-oxoamphotericins: insights into structure-activity relationships in polyene antibiotics[END_REF]. The co-incidental loss of two AmphC modules from strain KR10-1 raised the possibility that early termination might result from further unplanned deletions downstream of ACP11. This led us to characterise these strains more extensively. Here we show that the remaining PKS is intact. KR10 inactivation alone is sufficient to stall polyketide biosynthesis immediately after chain elongation by module 11.

Materials and methods

Growth of Streptomyces nodosus strains and spectrophotometric analysis of polyenyl-pyrones was carried out as described previously [START_REF] Murphy | Isolation and characterisation of amphotericin B analogues and truncated polyketide intermediates produced by genetic engineering of Streptomyces nodosus[END_REF].

Genomic DNA was purified using Qiagen Genomic-tip 500/G ion-exchange columns.

Oligonucleotide primers were obtained from MWG Biotech. Genome sequencing was carried out by BaseClear, Leiden, The Netherlands, using an Illumina Genome Analyser GAIIx. Direct sequencing of amplified DNA by the dideoxy method was carried out by Source BioScience, Ireland Sequencing Service, Dublin. PCR products were purified using a Qiagen QIAquick kit.

The Artemis programme (downloaded from http://www.sanger.ac.uk/resources/software/artemis/ ) was used for final assembly of the re-sequenced amphI region of strain KR10-1.

Results and discussion

Analysis of AmphI domains in KR10-1 and KR10-2 strains

In KR10-deficient mutants, module 11 incorporates a branched extender unit into the polyketide chain. Since this elongation stage proceeds normally, the lack of ketoreduction in cycle 11 was unexpected. To investigate whether KR11 was intact, the DNA specifying this domain was amplified from strains KR10-1 and KR10-2 and both PCR products were re-sequenced. The PCR primers KR11F and KR11R (Supplementary Table 1) were designed to amplify the regions coding for the N-and C-terminal interdomain linkers as well as the enzymatic domain. This sequence data showed that no mutations were present in the KR11 domains of either strain. To investigate whether deletions had occurred downstream of module 11, further PCR analysis was carried out on coding sequences for domains within downstream modules (ACP11, KS12, AT12, ACP12, KS13, AT13, KR13, ACP13, ACP14). (The primers used are listed in Supplementary Table 1.) Analysis of amplified DNA by agarose gel electrophoresis indicated that all coding sequences were present in KR10-1 and KR10-2 strains (Supplementary Fig. 1). More thorough analysis of strain KR10-1 was then carried out as described below.

Re-sequencing modules 11-14 in strain KR10-1

The AmphI protein contains modules 9 to 14 of the amphotericin PKS. The structures of the polyenyl-pyrones show that the KR10 PKSs function as expected up to module 10. As next-generation sequencing technology has become affordable, we used this approach to investigate more fully whether the downstream AmphI modules 11 to 14 remain intact. Our previous work has shown that targeted deletion of modules 5 and 6 leads to production of pentaene macrolactone analogues of amphotericin B in high yield [START_REF] Carmody | Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques[END_REF][START_REF] Murphy | Isolation and characterisation of amphotericin B analogues and truncated polyketide intermediates produced by genetic engineering of Streptomyces nodosus[END_REF]. This shows that deletion of these two modules is not relevant to early chain termination in KR10-1. However, this strain was selected for re-sequencing so that the spontaneous amphC deletion could be investigated as well as the modules downstream of KR10.

Genomic DNA from strain KR10-1 was sent to BaseClear for sequencing. De novo assembly provided 3977 contig sequences with an average size of 1818 nucleotides.

(In a separate study, we are currently sequencing and annotating the wild-type S. nodosus genome.) A second assembly of the strain KR10-1 sequence data was carried out using our unpublished S. nodosus genome sequence as a reference. This gave most of the strain KR10-1 amphI gene region coding for modules 11 to 14, although several small gaps remained. These regions were amplified by PCR (Supplementary Fig. 2) and directly sequenced by the dideoxy method. The KR10-1 coding sequence for AmphI modules 11 to 14 was re-sequenced completely. The sequence of this region of the protein (amino acid residues 3128 to 9510) was identical to that first published for the amphotericin PKS [START_REF] Caffrey | Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes[END_REF] with the following exception: R 6381 →A in ACP12. This difference is also present in the S. nodosus genome sequence and does not affect amphotericin production; it is thought to represent a neutral substitution or an error in the original sequence. Analysis of the KR10-1 genome indicated that the downstream AmphJ and AmphK PKS proteins were intact, although small gaps in the corresponding gene sequences were not closed. The re-sequencing also revealed the precise location of the deletion in the amphC gene (see below).

These results show that early termination occurs even though the programmed pathway specifies -ketoreduction by KR11 and further extension by downstream modules. There are no clear precedents where inactivation of a KR domain in one module prevents ketoreduction by the next. KR16 and KR12 mutants formed 3,5 diketoacyl and 3,5,7-triketoacyl intermediates, respectively, that are extended to form full length macrolactones [START_REF] Power | Engineered synthesis of 7-oxo-and 15-deoxy-15-oxoamphotericins: insights into structure-activity relationships in polyene antibiotics[END_REF]. The behaviour of the KR10 amphotericin PKSs is therefore surprising.

Proposed mechanisms for early termination in KR10-deficient PKSs

Module 11 normally generates a (2S, 3S)-2-methyl-3-hydroxyacyl-ACP product.

The landmark study of Weissman et al. (1997) first revealed the stereochemical aspects of such a cycle. A (2S)-methylmalonyl extender unit is loaded and incorporated, with inversion of stereochemistry, to give a (2R)-2-methyl-3-ketoacyl-ACP11. This undergoes epimerisation, via an enol intermediate, to give a (2S)-2-methyl-3-ketoacyl intermediate that is reduced to a (2S, 3S)-2-methyl-3-hydroxyacyl product. The epimerase activity has not been identified for any PKS module but is thought to reside within the KR domain [START_REF] Valenzano | The biochemical basis for sterochemical control in polyketide biosynthesis[END_REF]. In a reconstituted module lacking an epimerase, the C-2 proton of a (2R)-2-methyl-3-ketoacyl-thioester is protected by the ACP domain and is not exchangeable [START_REF] Castonguay | Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase[END_REF]. Our results suggest that in the unnatural tricarbonyl-ACP11 intermediate, the C-4 protons are labile and this allows tautomerisation of the C-5 ketone. The early enolisation stage of C-2 epimerisation then generates a dienol intermediate that is abnormally stable because it forms an extended system of conjugated double bonds. This is not a substrate for KR11 and is not accepted by KS12. This stalled polyketide intermediate might be off-loaded from the enzyme by the discrete AmphE thioesterase (TE) (Fig. 3, Route 1). These editing TEs mainly recognise decarboxylated extender units attached to ACP domains but can also remove non-extendable polyketide intermediates [START_REF] Heathcote | Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units[END_REF].

The linear acyl chain would form a terminal pyrone ring spontaneously. Alternatively, cis-enolisation of the tricarbonyl chain may favour concerted pyrone formation and thioester cleavage, thus bringing about efficient chain release without intervention of a TE (Fig. 3, Route 2). A cis geometry has been proposed for enolate intermediates that eventually form (2S 3S)-2-methyl-3-hydroxyacyl-ACP thioesters such as the product of AmphI module 11 (Starcevik et al., 2007).

With future work, it may still be possible to engineer the biosynthesis of 19deoxy-19-oxo-amphoteronolides. Mutations in module 11 that abolish the C-2 epimerase activity might allow reduction by KR11 and formation of full-length macrolactones with reversed alkyl group stereochemistry at C-16.

Iterative type I fatty acid and polyketide synthases are known to produce pyrones under abnormal conditions, such as the absence of modification enzymes or NADPH or S-adenosylmethionine co-factors. For example, incomplete lovastatin PKS systems synthesise a series of aberrant polyketides including polyenyl-pyrones of various chain lengths in vitro [START_REF] Ma | Complete reconstitution of a highly reducing iterative polyketide synthase[END_REF]. Inactivation of amphotericin PKS domains KR6, KR7, KR8 and KR9 would also result in formation of shorter polyenyl-3,5-diketoacyl intermediates. It seems unlikely that these would form non-extendable dienol intermediates because non-epimerising modules are thought to prevent loss of the C-2 proton from the -ketoacyl-ACP products. However, we plan to investigate this further by determining whether inactivation of KR7, KR8 or KR9 causes stalling of chain growth and accumulation of polyenyl-pyrones.

Mapping of the amphC deletion in strain KR10-1

This work also provides information on the evolution of natural product biosynthetic genes. In addition to finding that replacing a KR10 active site residue causes a drastic change in final product structure, we observed contraction of a heptaene synthase to a pentaene synthase. The genome re-sequencing suggested that the internal deletion in the KR10-1 amphC gene involves a 47-bp stretch that is directly repeated within the coding sequences for AT5 and AT7 (nucleotides 92165-92211 and 103628-103674, respectively, in sequence accession number AF357202). To confirm this, PCR primers M5F and M7R were designed to match AT5 and AT7 coding sequences flanking the putative deleted region. Direct sequencing of the product confirmed that the recombination had indeed generated an AT5/AT7 hybrid coding sequence, with the fusion point located within the repeated 47 bp sequence (Supplementary Fig 3). In a previous study [START_REF] Carmody | Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques[END_REF] Biotechnological potential of polyenyl-pyrone producer strains Numerous bioactive polyketides terminate with 2-pyrone groups that presumably protect linear chains from degradation by -oxidation [START_REF] Mcglacken | 2-Pyrone natural products and mimetics: isolation, characterisation and biological activity[END_REF].

Many of these compounds are biologically active and the polyenylpyrone group includes cytoprotectants, antioxidants, antibacterials and anticancer agents [START_REF] Clark | Biosynthesis of pyrrolylpolyenes in Auxarthron umbrinum Org[END_REF][START_REF] Coleman | Total synthesis of gymnoconjugatins A and B[END_REF]. The starter unit for these polyketides is critical for activity. The KR10-deficient amphotericin PKSs should also be useful for precursordirected biosynthesis of valuable analogues.

Conclusions

Further work will be required to elucidate fully the precise mechanism for early chain termination. However, the present results prove that inactivation of KR10 introduces a labile point at a later stage in the amphotericin PKS pathway. The production of tetraenyl-, pentaenyl-and heptaenyl-pyrones suggests that the corresponding domain may be critical in polyene PKSs in general. This work has uncovered a restriction on the theoretical number of polyene macrolactone analogues that can be generated by engineered biosynthesis.

Figure legends

  genetic engineering was used to construct a pentaene macrolactone synthase in which the C-terminal region of KS5 was fused to the interdomain linker preceding AT7. (The coding sequences were joined through KpnI sites representing nucleotides 91698-91703 and nucleotides 103146-103151 in sequence accession AF357202). Extensive screening was required to obtain this gene replacement mutant, indicating that this region does not form a recombination hot-spot. Since the amphotericin PKS genes are stable in wild-type S. nodosus, it is unclear why the amphC gene should undergo a spontaneous deletion in the KR10-1 strain.Recombination between directly repeated homologous sequences may have resulted by chance through incorrect repair of a double-strand break. Since 3, 4 and 5 are of no obvious benefit to the S. nodosus producer strains, continued propagation would presumably result in silencing or inactivation of these clusters by mutation. However, polyenylpyrone production remained stable when the KR10 strains were passaged through seven subcultures.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Structures of amphotericin B 1, 19-dideoxy-19-oxoamphoteronolide B 2, pentaenylpyrone 3, heptaenyl-pyrone 4 and tetraenyl pyrone 5.

Fig. 3 .

 3 Fig. 3. Proposed mechanisms for early chain termination in KR10 strains. Biosynthetic cycles 10 and 11 are shown. The -ketone formed by module 10 is unprocessed by the inactivated KR10 domain (shaded). Incorporation of a propionyl extender by module 11 gives a (2R)-2-methyl-3-ketoacyl-ACP11. This would be expected to undergo C-2 epimerisation and ketoreduction to give a (2S, 3S)-2-methyl-3-hydroxyacyl-chain that migrates from ACP11 to KS12. The first stage of C-2 epimerisation gives an enol intermediate that is unusually stable because the unnatural C5 ketone tautomerises to extend fully the system of conjugated double bonds. The resulting dienol intermediate is not reduced by KR11 or accepted by KS12. Two mechanisms for chain release are suggested. Route 1: Release by an editing TE gives a free acid that spontaneously isomerises and forms a pyrone. Route 2: Removal of the C-2 proton gives an intermediate containing a cis enol. This adopts a conformation that leads to concerted thioester cleavage and pyrone formation without intervention of a TE.

  Figure 1

  Figure 3

Acknowledgments

9

NK received a PhD studentship from the Irish Higher Education Authority Programme for Research in Third Level Institutions. PC received the financial support of Science Foundation Ireland under grant number 09/RFP/GEN2132.

Supplementary Table 1 Table 1. Oligonucleotides used in this work. AT12rsF3 and AT12rsR3 were used as primers for complete re-sequencing the PCR product shown in Supplementary Fig. 2 lane 9. AT14rsF2 and AT14rsR2 were used as primers for complete re-sequencing the PCR product shown in Supplementary Fig. 2 lane 7.

Supplementary data

Supplementary Fig. 1. PCR analysis of coding sequences for downstream modules in KR10 mutants. Reactions were carried out with primers designed from sequences coding for domains KS12 (lanes 1 to 4), AT12 (lanes 5 to 8), KS13 (lanes 9 to 12), AT13 (lanes 13 to 16) and KR13 (lanes 17 to 20). The DNA templates were from S. nodosus (lanes 1, 5, 9, 13 and 17), strain KR10-1 (lanes 2, 6, 10, 14, 18), strain 7,11,15,19) and a separate isolate of strain 8,12,16,20). All strains retained the downstream coding sequences. M 1 2 3 4 5 6 7 8 9 10 11 12 M 13 14 15 16 17 18 19 20 Supplementary Fig. 2 Amplification of amphI regions unrepresented in strain KR10-1 genomic sequence. PCR products were generated using primers listed in Table S2:

AT11rsF + AT11rsR (lane 1); AT12rsF + AT12rsR (lane 2); ACP12rsF + ACP12rsR (lane 3); AT13rsF + AT13rsR (lane 4); KR13rsF + KR13rsR (lane 5); KS14rsF + KS14rsR (lane 6); AT14rsF + AT14rsR (lane 7); KR14rsF + KR14rsR (lane 8);

AT12rsF2 + AT12rsF2 (lane 9). The PCR products were purified and directly sequenced by the dideoxy method.

M 1 2 3 4 5 6 7 8 9 10 3 2 1 0.5 kb Supplementary Fig. 3.

A.


Dr Gundula Behrens
email: gundula.behrens@klinik.uni-regensburg.de

Michael F Leitzmann

Sven Sandin

Marie Löf

Iris M Heid

Hans- Olov Adami

Elisabete Weiderpass

The association between alcohol consumption and mortality: the Swedish Women's Lifestyle and Health Study

Keywords: Alcohol, mortality, women's health, cohort study

Although light to moderate alcohol intake may reduce cardiovascular disease (CVD) mortality, the effect on total mortality requires further study, particularly among young and middle-aged women. We studied the association between alcohol consumption and mortality from all causes, from cancer, and from CVD in the Swedish Women's Lifestyle and Health Study, a cohort of 47,921 female residents of Sweden aged 30 to 49 years at baseline in 1991/1992 and followed up to 2006. We estimated the relative risk (RR) of mortality associated with alcohol intake using Cox regression adjusted for age, smoking, BMI, saturated fat intake, physical activity, and education. During 713,295 person-years of followup, 1,119 deaths occurred, including 158 deaths from CVD, 673 deaths from cancer, and 288 deaths from other causes. Compared with non-drinking, light to moderate drinking (0.1-19.9 grams of alcohol per day) showed a statistically significant inverse association with total mortality (RR=0.83, 95% CI=0.71-0.98). Analyses of cause-specific mortality revealed an RR for CVD mortality of 0.69 (95% CI=0.46-1.01) and an RR for cancer mortality of 0.92 (95% CI=0.75-1.15). These results suggest that in younger women, a possibly beneficial effect of light to moderate drinking on future risk of mortality is limited to a prevention of CVD mortality but not cancer mortality.

Introduction

Alcohol consumption plays an important role in numerous cultures, in particular in Western societies. The adverse effects of excessive alcohol consumption on health and mortality are well established [START_REF] Castelnuovo | Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies[END_REF]. It is also widely believed that modest alcohol consumption is beneficial to health [START_REF] Castelnuovo | Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies[END_REF]. However, the effects of alcohol on health may differ according to sex, age, and ethnicity [START_REF] Castelnuovo | Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies[END_REF][START_REF] Nakaya | Alcohol consumption and mortality in Japan: the Miyagi Cohort Study[END_REF][START_REF] White | Alcohol consumption and mortality: modelling risks for men and women at different ages[END_REF]. In addition, there has been concern about potential bias due to the mutual inclusion of lifetime abstainers, former heavy drinkers, and those who stopped consuming alcohol due to poor health into a single reference group of non-drinkers [START_REF] Nakaya | Alcohol consumption and mortality in Japan: the Miyagi Cohort Study[END_REF][START_REF] Klatsky | Wine, liquor, beer, and mortality[END_REF][START_REF] Dawson | Alcohol consumption, alcohol dependence, and all-cause mortality[END_REF]. A further concern is that light to moderate alcohol drinking may reduce risk of mortality from cardiovascular disease (CVD), in particular mortality from coronary heart disease (CHD) [START_REF] Kloner | To drink or not to drink? That is the question[END_REF][START_REF] Ikehara | Alcohol consumption and mortality from stroke and coronary heart disease among Japanese men and women: the Japan collaborative cohort study[END_REF], but that even modest intake of alcohol may adversely affect cancer mortality [START_REF] Breslow | Prospective study of alcohol consumption in the United States: quantity, frequency, and cause-specific mortality[END_REF][START_REF] Thun | Alcohol consumption and mortality among middle-aged and elderly U.S. adults[END_REF]. To further explore these issues, we examined the association between alcohol intake and mortality from any cause, CVD mortality, and cancer mortality in the Swedish Women's Lifestyle and Health Study with nearly 50,000 women living in Sweden. The cohort includes a large number of light to moderate drinkers, a level of alcohol intake that is considered controversial in terms of its potential health benefits. Furthermore, the cohort's baseline age range of 30 to 49 years is exceptional: previous studies have looked either at a much broader age range [START_REF] Thun | Alcohol consumption and mortality among middle-aged and elderly U.S. adults[END_REF][START_REF] Djousse | Alcohol consumption and risk of cardiovascular disease and death in women: potential mediating mechanisms[END_REF][START_REF] Lin | Alcohol consumption and mortality among middle-aged and elderly Japanese men and women[END_REF] or at older women [START_REF] Paganini-Hill | Type of alcohol consumed, changes in intake over time and mortality: the Leisure World Cohort Study[END_REF][START_REF] Simons | Moderate alcohol intake is associated with survival in the elderly: the Dubbo Study[END_REF].

Methods

The cohort

The Women's Lifestyle and Health (WLH) cohort is a prospective study of women that was approved by the Ethical Committee of the Karolinska Institute Stochholm and then initiated between August 1991 and June 1992 in the Uppsala Health Care Region, Sweden, when 96,000 female residents aged 30 to 49 years were randomly selected and posted a self-report questionnaire. Of these, 49,259 women gave their informed consent and were enrolled in the WLH study. A follow-up questionnaire was sent between February 2003 and January 2004, which a total of 34,402 women completed. Both questionnaires requested information regarding anthropometric measurements, personal history of diseases, smoking and drinking habits, diet, and physical activity.

Assessment of alcohol intake

On the baseline questionnaire, each woman was asked to report the number of glasses of beer (1 glass=2 dl), wine (1 glass=1dl), and spirits (1 glass=4 cl) that she currently drank per week, per month, or per year. The woman could also report that she never or seldom drank alcohol.

The reported quantities of beer, wine and spirits were converted to grams of alcohol using food composition data from the Swedish National Food Administration [START_REF]Food Composition Tables[END_REF]. The follow-up questionnaire contained a corresponding question regarding alcohol intake although the frequencies and quantities were somewhat differently formulated. The reported quantities of beer, wine and spirits were converted into quantities of alcohol in the same manner as those from the baseline questionnaire.

Analytic cohort

For the current analysis, we excluded 484 women with incomplete baseline information on alcohol or total energy intake, 984 women with extreme energy intakes (top or bottom 1% of the energy intake distribution), and 10 women who completed the baseline questionnaire but were later found to have emigrated from Sweden immediately afterwards, which left 47,921 women for our main analysis.

Endpoint definition

Cohort members were followed from August 1991 through December 31, 2006 by linking the cohort to the national registers (Death Register, Cancer Register, and Immigration Register) through their unique national registration numbers [START_REF] Kumle | Risk for invasive and borderline epithelial ovarian neoplasias following use of hormonal contraceptives: the Norwegian-Swedish Women's Lifestyle and Health Cohort Study[END_REF], making follow-up virtually complete [START_REF] Mattsson | Completeness of the Swedish Cancer Register. Non-notified cancer cases recorded on death certificates in 1978[END_REF][START_REF] Lunde | The person-number systems of Sweden, Norway, Denmark, and Israel[END_REF]. Cases of mortality were identified by the International Classification of Diseases codes (ICD-9 and ICD-10). The primary endpoint for the present analysis was mortality from any cause. In secondary analyses, we considered mortality from cancer (ICD-9 140-209 or ICD-10 C00-D09), CVD (ICD-9 390-459 or ICD-10 I00-I99), and non-cancer/non-CVD causes (ICD-9 001-139, 210-389, 460-999 or ICD-10 A00-B99, D10-H95, J00-Z99).

Statistical main analysis

Each study participant accrued follow-up time beginning at the date of return of the baseline questionnaire and ending at the date of death, emigration, or the end of follow-up in December 31, 2006, whichever occurred first. Following the Nurses' Health Study [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF] as closely as the number of cases allowed, alcohol intake categories were defined as 0, 0.1-1.4, 1.5-4.9, 5.0-9.9, 10.0-14.9, and 15.0+ grams of alcohol per day. For example, the category of 1.5-4.9 grams of alcohol per day corresponds to approximately two glasses of wine per week (1 glass = 1dl; alcohol by volume = 10%). To further facilitate comparison with previous studies, we also compared subjects with 0.1-19.9 grams of alcohol per day with subjects with no alcohol consumption [START_REF] Lin | Alcohol consumption and mortality among middle-aged and elderly Japanese men and women[END_REF][START_REF] Marugame | Patterns of alcohol drinking and all-cause mortality: results from a large-scale population-based cohort study in Japan[END_REF][START_REF] Baglietto | Average volume of alcohol consumed, type of beverage, drinking pattern and the risk of death from all causes[END_REF][START_REF] Hoffmeister | The relationship between alcohol consumption, health indicators and mortality in the German population[END_REF] as well as participants consuming any quantity of alcohol with non-drinkers [START_REF] Paganini-Hill | Type of alcohol consumed, changes in intake over time and mortality: the Leisure World Cohort Study[END_REF][START_REF] Simons | Moderate alcohol intake is associated with survival in the elderly: the Dubbo Study[END_REF][START_REF] Knoops | Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women: the HALE project[END_REF]. We fitted Cox proportional hazards regression models to estimate the relative risks of mortality associated with alcohol intake. We verified that the assumption of proportional hazards was not violated using Schoenfeld residuals [START_REF] Schoenfeld | Partial residuals for the proportional hazards regression model[END_REF].

For age-adjusted analyses, we used five-year age groups at baseline. In multivariable models, we additionally adjusted for body mass index (BMI) (<18.5, 18.5-24.9, 25.0+ kg/m 2 ), energyadjusted (24) saturated fat intake (grams per day in quintiles), smoking status (never, past, and current smoking with current intensities of 1-14, 15-24, 25+ cigarettes per day), level of physical activity [derived from the original self-assessment scale of 1 through 5 (very low to very high level of physical activity, considering both working and leisure time) as low (1 or 2), intermediate [START_REF] White | Alcohol consumption and mortality: modelling risks for men and women at different ages[END_REF], or high (4 or 5)], and education level (0-10, 11-13, 14+ years). For tests of linear trend across categories of alcohol intake, we assigned each categorical alcohol value the median alcohol intake within that category and determined the P value of the resulting continuous variable using the Wald test.

All relative risks (RRs) are presented with 95% confidence intervals (CI), and reported P values are based on two-sided tests at the 5% significance level. All analyses were conducted using the statistical software SAS release 9.2 (SAS Institute, Cary, NC).

Statistical sub-analyses

Firstly, to assess potential bias by preclinical chronic disease, we repeated the analysis excluding all women with pre-existing hypertension, type 2 diabetes mellitus, or cancer and additionally excluded the first two years of follow-up for all women. Secondly, to investigate consistent drinking patterns we used data on consistent drinking versus consistent alcohol avoidance from both the baseline and the 2003 follow-up questionnaires, restricting the analysis to deaths that occurred between 2003 and 2006. Thirdly, we addressed potential confounding due to including past heavy drinkers in the reference group of non-drinkers by excluding non-drinkers in 2003 who had previously reported alcohol intake of 10 grams per day or more in 1991/1992. Fourthly, to examine whether the association of alcohol to mortality was modified by other risk factors for mortality, we conducted tests for multiplicative interaction using likelihood-ratio tests. We also evaluated the relation of alcohol to mortality within strata of potential effect modifying variables. Fifthly, we performed mortality type specific analyses analogously to the all-cause mortality investigations.

Finally, we conducted beverage type specific analyses for wine, beer and spirits, classified as 0, 0.1-1.4, 1.5-4.9, or 5.0+ grams per day. These analyses were mutually adjusted for alcohol intake from other beverage types, e.g. the wine analysis was adjusted for beer and spirits intakes. Of the 47,921 women in our analytic cohort, 13.4% declared no alcohol intake, the majority of women (84.5%) reported alcohol intakes of 0.1 to 14.9 grams per day, and 2.1% of participants stated an average daily alcohol intake of 15 grams or more at baseline. The vast majority (over 99%) of women consumed less than 20 grams of alcohol per day. In the intake groups of 0, 0.1-1.4, 1.5-4.9, 5.0-9.9, and 10.0-14.9 grams of alcohol per day, deaths were mainly attributed to breast cancer (14-19% of all deaths) or to cancers of the trachea, bronchus or lung (9-13%). By comparison, in the highest intake group of 15.0+ grams of alcohol or more per day, the top two causes of mortality were injury/poisoning (12%) and breast cancer (8%). The correlation coefficient between wine and beer intake was 0.11, between wine and spirits intake it was 0.15, and between beer and spirits intake it was 0.16, implying that study participants showed no strong preference for the combination of any two alcoholic beverage types.

Results

Distribution of events and alcohol intake

Distribution of risk factors at baseline

We present summary statistics of potentially confounding factors by categories of total alcohol intake (Table 1). With increasing total alcohol intake, women were more likely to be past or current smokers, which would tend to increase the risk of mortality. On the other hand, women with greater total alcohol consumption were better educated, they showed less saturated fat intake, and they were less likely to have been diagnosed with diabetes than women who abstained from alcohol, a risk factor profile that would tend to decrease risk of mortality. Physical activity, which also tends to decrease mortality risk, was highest among women in the intermediate alcohol categories between 1.5 and 14.9 grams per day. BMI and history of hypertension did not vary appreciably according to alcohol intake. Corresponding figures for baseline characteristics by specific alcoholic beverage types can be found in the (web-based) Supplementary Table 1.

All-cause mortality

We investigated the relation of total alcohol intake to all-cause mortality using the zero intake group as reference (Fig. 1). In multivariable analyses, we inspected the mortality risk in the intake groups 0.1-1.4, 1.5-4.9, 5.0-9.9, 10.0-14.9, and 15.0+ grams of alcohol per day compared to alcohol abstinence and found null associations for all levels with the exception of one category: for 1.5-4.9 grams of alcohol per day the association proved to be statistically significant and inverse (RR=0.81, 95% CI=0.67-0.97). Statistically significant inverse associations were also found for the larger group of 0.1-19.9 grams of alcohol intake per day (RR=0.83, 95% CI=0.71-0.98) versus none, and for any versus no alcohol intake (RR=0.84, 95%=0.71-0.98). Adjusting for hypertension status and diabetes status did not appreciably affect the results (data not shown). Excluding women with a history of hypertension, type 2 diabetes mellitus, or cancer at baseline and excluding the first two years of follow-up did not essentially alter the results (data not shown). In 2003, 10.3% of women were non-drinkers, 80.7% of women stated an alcohol intake of 0.1 to 14.9 grams per day, and 9.0% of women consumed over 15 grams of alcohol per day. Over 95% of women reported an alcohol intake less than 20 grams of alcohol per day in 2003. Age-adjusted analyses revealed that women consistently reporting alcohol use in 1991/1992 and in 2003 had a statistically significant lower mortality risk between 2003 and 2006 than consistent non-drinkers (RR=0.64, 95% CI=0.41-1.00). Exclusion of potential former heavy drinkers from the non-drinking group did not alter those results.

Cause-specific mortality

We examined the relation between alcohol intake and mortality from specific causes (Table 2). In analyses adjusted for potential confounding factors, we observed an RR for CVD mortality of 0.69 (95% CI=0.46-1.01) comparing an alcohol intake of 0.1-19.9 grams per day with no alcohol intake and an RR for cancer mortality of 0.92 (95% CI=0.75-1.15) comparing an alcohol intake of 0.1-19.9 grams per day with no alcohol intake.

Analyses within strata of mortality risk factors

In multivariable analyses, we looked at associations between alcohol and mortality within strata of other risk factors (Table 3). In women aged 30 to 34 years at baseline, the highest intake group of 15 grams of alcohol per day or more revealed a statistically significant increase in total mortality risk (RR=3.11, 95% CI=1.18-8.20) compared to no alcohol intake, while the intermediate intake levels showed null associations. In contrast, among women aged 35 to 49 years at baseline the highest intake group of more than 15 grams of alcohol per day showed the strongest, statistically significant risk reduction (RR=0.55, 95% CI=0.34-0.90) compared to non-drinkers. In that age group, intake levels 0.1-1.4 grams of alcohol per day (RR=0.81, 95% CI=0.67-0.99) and 1.5-4.9 grams of alcohol per day (RR=0.76, 95% CI=0.63-0.92) were statistically significantly and inversely associated with mortality (P value for interaction by age=0.047). The association between alcohol and mortality did not vary across strata defined by BMI, saturated fat intake, smoking, history of hypertension, type 2 diabetes mellitus, physical activity, or education (data not shown).

Specific alcoholic beverage types

We also performed analyses by specific alcoholic beverage type (Table 4). The only statistically significant results from the multivariable analyses were as follows: compared to zero beer intake, 0.1-1.4 grams of alcohol per day from beer intake (RR=0.83, 95% CI=0.72-0.96) and 1.5-4.9 grams of alcohol per day from beer intake (RR=0.83, 95% CI=0.70-0.98) were inversely associated with total mortality. In contrast, we noted a statistically significant positive association for women with an average daily alcohol from spirits intake of 5 grams or more (RR=1.52, 95% CI=1.04-2.22, p-trend=0.05).

Discussion

In this cohort of mostly light to moderate drinking women aged 30 to 49 years at baseline compared to non-drinkers, we observed an approximately 20 to 30% apparent reduction in mortality risk for various categories of alcohol intake within the range of 1.5 to 20 grams per day, with an overall 16% reduction in risk for mortality when we compared women with any versus no alcohol intake. We were unable to assess the association with alcohol intake beyond 20 grams per day because of insufficient numbers of women (1% of women in our cohort).

A study from England and Wales revealed that as age increases, the level of alcohol intake associated with maximal risk reduction increases as well, suggesting that results from cohorts with different age distributions cannot be strictly compared [START_REF] White | Alcohol consumption and mortality: modelling risks for men and women at different ages[END_REF]. The study most closely related to ours with respect to its age and sex structure and alcohol and mortality results is the Nurses' Health Study with an analytic sample of 86,000 women aged 35 to 59 years at baseline [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF]. That study revealed a statistically significant inverse relation of alcohol intake to mortality for an intake range of 1.5 to 14.9 grams per day (RR for 1.5 to 4.9 grams per day=0.83; 95% CI=0.74-0.93; RR for 5.0 to 14.9 grams per day=0.88; 95% CI=0.79-0.99) with estimates similar to ours. Studies with wider age ranges than ours or with partly overlapping age structures also detected statistically significant inverse (9-11, 25, 26) or null [START_REF] Marugame | Patterns of alcohol drinking and all-cause mortality: results from a large-scale population-based cohort study in Japan[END_REF][START_REF] Baglietto | Average volume of alcohol consumed, type of beverage, drinking pattern and the risk of death from all causes[END_REF][START_REF] Hoffmeister | The relationship between alcohol consumption, health indicators and mortality in the German population[END_REF] associations between alcohol intakes of up to 20 grams per day and all-cause mortality. Studies in women over age 50 years also found statistically significant mortality risk reductions associated with any versus no alcohol intake [START_REF] Paganini-Hill | Type of alcohol consumed, changes in intake over time and mortality: the Leisure World Cohort Study[END_REF][START_REF] Simons | Moderate alcohol intake is associated with survival in the elderly: the Dubbo Study[END_REF]. One combined analysis in men and women over age 50 years confirmed a statistically significant risk reduction for any versus no alcohol intake [START_REF] Knoops | Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women: the HALE project[END_REF], another detected statistically significant risk reductions for alcohol intakes of up to 28 grams per day only [START_REF] Gronbaek | Alcohol and mortality: is there a U-shaped relation in elderly people?[END_REF].

In the youngest women in our cohort, i.e., in those aged 30 to 34 years at baseline, alcohol intake of 15 grams or more per day was associated with increased mortality risk, whereas in women aged 35 years or older at baseline alcohol consumption of 15 grams per day of alcohol or more was related to decreased mortality risk. The Nurses' Health Study also found that for any given level of alcohol intake, the apparent adverse effect of alcohol on risk for mortality was consistently more pronounced among younger than older women [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF], ranging from RR=2.46 (95% CI=0.50-12.4) for women aged 34 to 39 years to RR=1.02 (95% CI=0.80-1.30) for women over 60. Similar conclusions were reached by the Japanese Collaborative Cohort Study [START_REF] Lin | Alcohol consumption and mortality among middle-aged and elderly Japanese men and women[END_REF][START_REF] Rehm | Alcohol consumption and all-cause mortality[END_REF] and the National Health Epidemiology Follow-up Study [START_REF] Lin | Alcohol consumption and mortality among middle-aged and elderly Japanese men and women[END_REF][START_REF] Rehm | Alcohol consumption and all-cause mortality[END_REF].

In our study, a null association was observed between alcohol consumption and cancer mortality. Similarly, the Nurses' Health Study detected a null association for cancer mortality with alcohol intake up to 30 grams per day [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF][START_REF] Van Dam | Combined impact of lifestyle factors on mortality: prospective cohort study in US women[END_REF]. A modest positive association was observed for intake levels of 30 grams per day or more in the Nurses' Health Study [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF][START_REF] Van Dam | Combined impact of lifestyle factors on mortality: prospective cohort study in US women[END_REF].

Other studies showed statistically significant positive [START_REF] Klatsky | Wine, liquor, beer, and mortality[END_REF][START_REF] Breslow | Prospective study of alcohol consumption in the United States: quantity, frequency, and cause-specific mortality[END_REF][START_REF] Thun | Alcohol consumption and mortality among middle-aged and elderly U.S. adults[END_REF] or null [START_REF] Lin | Alcohol consumption and mortality among middle-aged and elderly Japanese men and women[END_REF] associations between alcohol and cancer mortality in women or in men and women combined [START_REF] Gronbaek | Type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer[END_REF].

We noted an inverse association between low alcohol intake (1.5 to 4.9 grams and 0.1-19.9 grams of alcohol per day) and mortality from CVD. The Nurses' Health Study observed statistically significant decreased risks of CVD mortality with 1.5 to 4.9 grams (RR=0.57; 95% CI=0.43-0.76), 5.0 to 14.9 grams (RR=0.73; 95% CI=0.56-0.95), and 15.0 to 29.9 (RR=0.66; 95% CI=0.47-0.93) grams of alcohol intake per day [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF][START_REF] Van Dam | Combined impact of lifestyle factors on mortality: prospective cohort study in US women[END_REF]. Other studies also revealed inverse relations between alcohol intake and CVD (or CHD) mortality in women [START_REF] Thun | Alcohol consumption and mortality among middle-aged and elderly U.S. adults[END_REF][START_REF] Theobald | The effects of alcohol consumption on mortality and morbidity: a 26-year follow-up study[END_REF][START_REF] Mukamal | Alcohol consumption and cardiovascular mortality among U.S. adults, 1987 to 2002[END_REF][START_REF] Klatsky | Risk of cardiovascular mortality in alcohol drinkers, ex-drinkers and nondrinkers[END_REF] or in men and women combined [START_REF] Knoops | Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women: the HALE project[END_REF][START_REF] Gronbaek | Type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer[END_REF]. Non-linear inverse associations for low alcohol intakes and positive [START_REF] Rehm | Alcohol consumption and coronary heart disease morbidity and mortality[END_REF] or null (4, 7, 8, 10) associations for higher alcohol intakes were also identified in numerous studies among women.

Similar to our study, most other studies found an inverse association between beer consumption and mortality in women [START_REF] Paganini-Hill | Type of alcohol consumed, changes in intake over time and mortality: the Leisure World Cohort Study[END_REF][START_REF] Simons | Moderate alcohol intake is associated with survival in the elderly: the Dubbo Study[END_REF][START_REF] Lapidus | Alcohol intake among women and its relationship to diabetes incidence and all-cause mortality: the 32-year follow-up of a population study of women in Gothenburg, Sweden[END_REF] or in men and women combined [START_REF] Gronbaek | Type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer[END_REF][START_REF] Klatsky | Risk of cardiovascular mortality in alcohol drinkers, ex-drinkers and nondrinkers[END_REF].

Only few studies reported a positive relation of beer to mortality [START_REF] Baglietto | Average volume of alcohol consumed, type of beverage, drinking pattern and the risk of death from all causes[END_REF][START_REF] Harriss | Alcohol consumption and cardiovascular mortality accounting for possible misclassification of intake: 11-year follow-up of the Melbourne Collaborative Cohort Study[END_REF]. Inverse associations between wine intake and mortality risk were noted in studies among women [START_REF] Klatsky | Wine, liquor, beer, and mortality[END_REF][START_REF] Paganini-Hill | Type of alcohol consumed, changes in intake over time and mortality: the Leisure World Cohort Study[END_REF][START_REF] Baglietto | Average volume of alcohol consumed, type of beverage, drinking pattern and the risk of death from all causes[END_REF][START_REF] Lapidus | Alcohol intake among women and its relationship to diabetes incidence and all-cause mortality: the 32-year follow-up of a population study of women in Gothenburg, Sweden[END_REF][START_REF] Harriss | Alcohol consumption and cardiovascular mortality accounting for possible misclassification of intake: 11-year follow-up of the Melbourne Collaborative Cohort Study[END_REF] or in analyses involving both women and men [START_REF] Gronbaek | Type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer[END_REF][START_REF] Klatsky | Risk of cardiovascular mortality in alcohol drinkers, ex-drinkers and nondrinkers[END_REF]. We observed a positive association between spirits intake and risk of mortality. Previous studies reported inverse [START_REF] Paganini-Hill | Type of alcohol consumed, changes in intake over time and mortality: the Leisure World Cohort Study[END_REF], null [START_REF] Baglietto | Average volume of alcohol consumed, type of beverage, drinking pattern and the risk of death from all causes[END_REF][START_REF] Lapidus | Alcohol intake among women and its relationship to diabetes incidence and all-cause mortality: the 32-year follow-up of a population study of women in Gothenburg, Sweden[END_REF], or positive [START_REF] Harriss | Alcohol consumption and cardiovascular mortality accounting for possible misclassification of intake: 11-year follow-up of the Melbourne Collaborative Cohort Study[END_REF] relations between spirits intake and mortality risk. Previous analyses combining men and women showed null [START_REF] Klatsky | Risk of cardiovascular mortality in alcohol drinkers, ex-drinkers and nondrinkers[END_REF] or positive [START_REF] Gronbaek | Type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer[END_REF][START_REF] Gronbaek | Mortality associated with moderate intakes of wine, beer, or spirits[END_REF] associations.

One of our study's strengths is its particular age and sex structure of younger women. Further merits are that potential recall bias was precluded by the prospective study design, potential information bias was minimized by the reasonably valid and reliable food frequency questionnaire comparable to that used in a similar study [START_REF] Wolk | A prospective study of association of monounsaturated fat and other types of fat with risk of breast cancer[END_REF], and potential selection bias was avoided by the virtually complete follow-up. In addition, we minimised the potential for confounding by controlling for numerous accepted risk factors for mortality. Since previous studies reported both greater [START_REF] Nakaya | Alcohol consumption and mortality in Japan: the Miyagi Cohort Study[END_REF][START_REF] Klatsky | Wine, liquor, beer, and mortality[END_REF][START_REF] Dawson | Alcohol consumption, alcohol dependence, and all-cause mortality[END_REF][START_REF] Liao | Alcohol intake and mortality: findings from the National Health Interview Surveys (1988 and 1990)[END_REF][START_REF] Harriss | Alcohol consumption and cardiovascular mortality accounting for possible misclassification of intake: 11-year follow-up of the Melbourne Collaborative Cohort Study[END_REF][START_REF] Freiberg | Alcohol consumption, hypertension, and total mortality among women[END_REF][START_REF] Cullen | Alcohol and mortality in Busselton, Western Australia[END_REF] or lower mortality risk [START_REF] Lin | Alcohol consumption and mortality among middle-aged and elderly Japanese men and women[END_REF][START_REF] Baglietto | Average volume of alcohol consumed, type of beverage, drinking pattern and the risk of death from all causes[END_REF] among female former drinkers compared to female lifetime abstainers, we were concerned with potential bias through the inclusion of ill former heavy drinkers in the reference group, but found that the exclusion of former drinkers did not alter our results. Our study had limited power to explore associations with specific causes of mortality and mortality among women with alcohol intakes with over 20 grams per day.

Moderate intake of ethanol could prevent CVD by numerous biological mechanisms, such as improving endothelial function, lowering blood pressure, reducing inflammation, increasing high density lipoprotein (HDL) cholesterol and decreasing low density lipoprotein (LDL) cholesterol, enhancing fibrinolysis, inhibiting platelet aggregation and coagulation, attenuating insulin resistance, and releasing stress [START_REF] Kloner | To drink or not to drink? That is the question[END_REF][START_REF] Gronbaek | The positive and negative health effects of alcohol-and the public health implications[END_REF][START_REF] O'keefe | Alcohol and cardiovascular health: the razor-sharp double-edged sword[END_REF]. In addition to ethanol, other components of alcoholic beverages, thus as flavonoids and resveratrol, prevent CVD in ways similar to ethanol ( 6). An open issue is whether the apparent benefit of ethanol for vascular health depends on alcoholic beverage type. A meta-analysis reported inverse relations of both wine and beer intake to CVD risk that was greater for wine than for beer intake [START_REF] Castelnuovo | Meta-analysis of wine and beer consumption in relation to vascular risk[END_REF].

Divergent beverage type-specific results might arise due to residual confounding by sociodemographic or lifestyle factors; the greatest risk reductions were typically observed for the alcoholic beverage type predominantly consumed in the specific population under study [START_REF] Klatsky | Wine, liquor, beer, and mortality[END_REF][START_REF] Rimm | Review of moderate alcohol consumption and reduced risk of coronary heart disease: is the effect due to beer, wine, or spirits[END_REF].

In conclusion, we observed a moderate reduction in total and CVD mortality but not in cancer mortality among women with any versus no alcohol intake. These results, broadly consistent with findings from previous studies, suggest a beneficial effect of light to moderate drinking on women's health. Despite the apparent benefits of moderate alcohol intake for mortality risk, health care providers should discuss the potential hazards of alcohol consumption with their patients and provide them with an individual clinical recommendation. years, where ten years of education correspond to completion of compulsory school, 13 years of education correspond to completion of secondary school, and 14 years of education or more correspond to graduate study at university). The alcohol-type specific analyses (wine/beer/spirits) in grams of alcohol per day are additionally adjusted for the intake of complementary alcohol types (0, 0.1-1.4, 1.5-4.9, 5.0+ grams/day), e.g. the wine analysis is adjusted for beer and for spirits intakes, while the alcohol-type specific analyses (wine/beer/spirits) in per cent of total alcohol intake are additionally adjusted for the level of total alcohol intake (0, 0.1-1.4, 1.5-4.9, 5.0-9.9,10.0-14.9,15+ grams/day). c Adjusted for age (5-year categories), BMI (<18.5, 18.5-24.9, 25.0+ kg/m²), energy-adjusted saturated fat intake (quintiles), smoking status (never, past, and current smoking with current intensities of 1-14 cigs/day, 15-24 cigs/day, and 25+ cigs/day), current physical activity (low, intermediate, high), years of education (0-10, 11-13, 14+ years, where ten years of education correspond to completion of compulsory school, 13 years of education correspond to completion of secondary school, and 14 years of education or more correspond to graduate study at university). The alcohol-type specific analyses (wine/beer/spirits) in grams of alcohol per day are additionally adjusted for the intake of complementary alcohol types (0, 0.1-1.4, 1.5-4.9, 5.0+ grams/day), e.g. the wine analysis is adjusted for beer and for spirits intakes.

  During 713,295 person-years of follow-up between 1991 and 2006, a total of 1,119 deaths occurred, including 673 deaths from cancer (184 from breast cancer, 122 from cancers of the trachea, bronchus, and lung, and 60 from ovarian cancer), 158 from CVD (69 from CHD, 12 from stroke, and 77 from other CVD-related causes), 54 deaths from injury or poisoning, 39 deaths from suicide, and 195 deaths from other causes. Censoring events were distributed between 7 days and 15.3 years after study entry, with a mean censoring time of 15.0 years. Deaths occurred between 68 days and 15.3 years after study entry with a mean of time to death of 9.2 years. Thirty subjects died and 79 subjects were censored within one year of entry. The mean ages at baseline and at the end of follow-up were 39.2 years and 54.3 years, respectively.

Table 1 .

 1 Age-standardized a baseline characteristics according to alcohol intake among women in the Swedish Women's Lifestyle and Health Study (Aug 1991 -Jun 1992) Relative Risk of Mortality from any Cause in Relation to Average Daily Total Alcohol Intake Among Swedish Women: The Swedish Women's Lifestyle and Health Study, 1991-2006. The age-adjusted relative risks were: RR for zero alcohol intake=1 (ref.); RR for 0.1-1.4

		Total Alcohol Intake (g/day)
	0	0.1-1.4	1.5-4.9	5.0-9.9	10.0-14.9	15.0+

Table 2 .

 2 Relative Risk of Cause-specific Mortality in Relation to Average Daily Total Alcohol Intake Among Swedish Women: The Swedish Women's Lifestyle and HealthStudy, 1991Study, -2006. .

	Relative Risk (95% CI) a

Table 3 .

 3 Multivariable Relative Risks of Total Mortality in Relation to Average Daily Total Alcohol Intake, Stratified by Age, Among Swedish Women: The Swedish Women's Lifestyle and Health Study, 1991-2006. high), years of education (0-10, 11-13, 14+ years, where ten years of education correspond to completion of compulsory school, 13 years of education correspond to completion of secondary school, and 14 years of education or more correspond to graduate study at university).

	Multivariable-adjusted

a CI=confidence interval; relative risks adjusted for age (5-year categories), BMI (<18.5, 18.5-24.9, 25.0+ kg/m²), energy-adjusted saturated fat intake (quintiles), smoking status (never, past, and current smoking with current intensities of 1-14 cigs/day, 15-24 cigs/day, and 25+ cigs/day), current physical activity (low, intermediate,

Table 4 .

 4 Relative Risk of Mortality from any Cause in Relation to Average Daily Beverage-type-specific Alcohol Intake Among Swedish Women: The Swedish Women's Lifestyle and HealthStudy, 1991Study, -2006. .

	Relative Risk (95% CI) a

a CI=confidence interval b

Acknowledgments

We thank Pouran Almstedt for excellent data management.