From a process perspective, improvements in plant design can lower the production cost, providing better profitability and competitiveness if the conversion of the whole sugar cane is considered. Simulations have been performed with AspenPlus to investigate how process integration can affect the minimum ethanol selling price of this 2G process (MESP-2G), as well as improve the plant energy efficiency. This is achieved by integrating the well-established sucrose-to-bioethanol process with the enzymatic process for lignocellulosic materials. Bagasse and leaves were steam pretreated using HThe addition of a steam dryer, doubling of the enzyme dosage in enzymatic hydrolysis, including leaves as raw material in the 2G process, heat integration and the use of more energy-efficient equipment led to a 37 % reduction in MESP-2G compared to the Base case. Modelling showed that the MESP for 2G ethanol was 0.97 US$/L, while in the future it could be reduced to 0.78 US$/L. In this case the overall production cost of 1G + 2G ethanol would be about 0.40 US$/L with an output of 102 L/ton dry sugar cane including 50 % leaves. Sensitivity analysis of the future scenario showed that a 50 % decrease in the cost of enzymes, electricity or leaves would lower the MESP-2G by about 20%, 10% and 4.5%, respectively.According to the simulations, the production of 2G bioethanol from sugar cane bagasse and leaves in Brazil is already competitive (without subsidies) with 1G starch-based bioethanol production in Europe. Moreover 2G bioethanol could be produced at a lower cost if subsidies were used to compensate for the opportunity cost from the sale of excess electricity and if the cost of enzymes continues to fall. Currently in Brazil, the production of sugar cane bioethanol is based entirely on the fermentation of sugar juice from sugar cane and/or molasses in autonomous distilleries (39% of the cases) and in plants associated with sugar mills 61%) [% [1]. ThThe view of bagasse has changed throughout the years, due to technological breakthroughs, investment opportunities and revenue margin. At the beginning of the Pro-álcool project in the 1970s, bagasse was considered a fluffy waste which was eliminated in low-efficiency 22- bar boilers. As a result of deregulation of the electricity market and the increase in the price of electricity due to the 2001 energy crisis , bagasseSugar cane leaves and tops, often called trash, constitute the residues of mechanical harvesting, and are suitable as raw material for 2G bioethanol production because of their lignocellulosic nature. The amount of trash that can be removed from the field without affecting the nutritional properties of the soil or pest and weed control is generally considered to be about 50% by weight , althougGenerally, the main process bottlenecks in 2G ethanol production, such as the low yield from the conversion of lignocellulose into fermentable sugars and the downstream processing, account for a significant proportion of the production cost . SeveralAmong the viable processes, steam pretreatment followed by enzymatic hydrolysis is one of the most promising approaches for ethanol production from lignocellulose , and thiProcess design and optimization greatly affect the production cost. The choice between simultaneous saccharification and fermentation and SHF, which have different advantages and disadvantages regarding their effects on downstream processing ; boiler Apart from technical hurdles and the price of petrol, the question of feedstock competitiveness must be addressed at international and local levels. The global price of sugar and the local bioelectricity price may affect 1G and 2G ethanol production, respectively. Although there is no direct competition with food security and supply, 2G ethanol from sugar cane suffers from a higher production cost due to the rise in opportunity cost caused by the rapidly growing bioelectricity market in Brazil. Today, greater profit may be achieved by burning bagasse and leaves than by producing 2G ethanol due to a lack of cheap fuel to complement hydropower in the dry season. However, future development of the 2G ethanol process using leaves and bagasse is recognized to be profitable, even when a high surplus of electricity (184 kWh/ton SC) is sold . In addi# [In the present study, a techno-economic evaluation of integrated 1G and 2G ethanol production from sugar cane has been performed for fifteen scenarios. In each scenario several operating conditions were implemented in the flowsheeting models, based on data obtained experimentally within the CaneBioFuel Project # , and difThe integration of 1G and 2G ethanol production was carried out sequentially by first investigating various process parameters, e.g. water insoluble solids (WIS), enzyme dosage and residence time in the enzymatic hydrolysis (EH), and then choosing the best conditions to evaluate the effect of various levels of integration. Each scenario was assessed by calculating the energy efficiency, and the running and capital costs, all of which have a significant effect on the MESP-2G ethanol. Excluding the Reference scenario, fourteen scenarios of the combined 1G + 2G process (A-L2) were simulated according to the EH operating conditions and process configurations summarized in Table First, a Reference scenario was simulated for the 1G process  represents the combined 1G + 2G plant, to evaluate the effects on the 2G process of changing the operating conditions and level of process integration between 1G and 2G ethanol production, without mixing the material streams. The schematic flowsheet for the Base case is shown in Figure An overview of costs for the simulated scenarios for 1G + 2G process can be found in Table Scenario B consists of the Base case (Scenario A) with a steam dryer for the EH solid residues to increase the lower heating value of the fuel by the removal of water, from 35 wt- % to 80 wt-% dry matter (DM). The steam dryer was assumed to work at 20 bar with superheated steam as drying medium. The effect of this in terms of energy is that more heat is available for steam and electricity production due to the reduction in heat loss by water vapour leaving with the flue gases. Therefore, more bagasse is available for ethanol production, but still not the full amount (about 94%). The water removal efficiency of the dryer is better than 85%, calculated from the energy recovery in the secondary steam. The steam dryer increases the heat use by 3.5% resulting in a process energy efficiency of 61.4%. From the cost point of view, the MESP-2G is lower than in Scenario A, indicating that including a dryer paid off. The drying unit was therefore included in all the following scenarios due to the beneficial effects in terms of energy and cost.Scenario B was then varied, mainly by changing the operating conditions in the EH step such as the enzyme dosage  and the residence time .The residence time in EH is a key factor that affects the conversion yield and capital cost directly, and the energy and ethanol production cost indirectly. A short residence time results in both a reduction in sugar yield and a reduction in the capital cost for the EH tanks. In Scenario C the residence time was shortened by 24 h compared with Scenario B, i.e. from 72 to 48 hours. It was still necessary to use a fraction of the bagasse (2%) for combined heat and power production. The main effect was a reduction in the ethanol production, from 57 to 53 L/ton-dry SC (ton-dSC), while there was no variation from the energy efficiency point of view (61.4%). However, the MESP-2G for Scenario C is definitely higher than for Scenario B, so the savings in the capital cost of EH did not balance the decrease in revenue from selling ethanol at the enzyme loading investigated.Enzyme loading was changed in the various scenarios to assess the impact of enzyme cost per litre of ethanol produced. In Scenario D the enzyme dosage was double that in Scenario B to investigate if a higher sugar yield and concentration could compensate for the increased cost of enzymes. No other modifications were applied to the flowsheet. As the amount of ethanol produced per ton of bagasse increased, less solid residue was available for CHP generation, and 16.3% of the total bagasse had to be used to ensure self-sufficiency in steam. Due to the higher EH yield, the final ethanol concentration prior to distillation also increased, reducing the energy demand as well as the capital cost for the distillation stage. This is also confirmed by the increase in energy efficiency for Scenario D to 62.0%. Although the total ethanol produced increased by exactly 1/3, the equipment cost did not increase correspondingly. However, the most positive effect of doubling the enzyme dosage was the decrease in the MESP-2G from 1.36 to 1.20 US$/L ethanol.A similar comparison was performed between Scenarios C and B to determine whether lowering the residence time had positive effects on the MESP-2G, but at higher enzyme loadings in EH. In Scenario E the enzyme dosage was the same as in Scenario D, while the EH residence time was 48 h instead of 72 h. The amount of bagasse required for CHP production in this scenario was 9.8% of the total amount, so the amount of bagasse available for 2G ethanol was 90.2%. Also in this case, a decrease in residence time did not pay off in spite of higher enzyme loading, and the MESP-2G increased compared to that for Scenario D.Among Scenarios A to E, Scenario D showed the lowest MESP-2G, and it was therefore used as the basis for the addition of leaves (Scenario F) and for heat integration of the combined 1G + 2G process (Scenario G). Stream integration was simulated by combining the distillation streams from the 1G and 2G processes in the same unit  which is non-integrated. As a matter of fact, the energy efficiency reached 64.7%. The amount of steam saved allowed the use of all the bagasse for ethanol production, which increased from 76 L/ton-dSC in Scenario D to 95 L/ton-dSC in Scenario G. Consequently, the MESP-2G also decreased, from 1.20 to 1.16 US$/L.Scenario I  consists of blending part of the liquid after EH of bagasse with the sugar juice from cane before and after evaporation in order to obtain an intermediate concentration of 25 wt-% sucrose and a final total concentration of mixed sugars of 8.5 wt-%, as shown in Figure The addition of leaves was also evaluated in the integrated Scenarios G and J, resulting in Scenarios H and K1, respectively, where EH of leaves was performed at 14% WIS. In Scenario K2 the EH of leaves was performed at 7% WIS, which is the only difference compared with Scenario K1 . Both Scenarios F and K2 showed the highest 2G ethanol production (151 L/ton-dSC) and yield.Despite the fact that Scenario F includes no integration , the MESP-2G obtained, 0.97 US$/L, is the lowest of all the scenarios investigated. The high level of integration of Scenario K2 (heat and streams), already described in Scenario J, has a greater effect on the energy savings than Scenario F (5 MW). However, the capital cost in Scenario K2 increased to 334 million US$, compared with 299 million US$ in Scenario F. This negative impact on the total production cost for Scenario K2 is greater than the positive effect of the revenue obtained from the 16% increase in the amount of electricity produced, resulting in a slightly higher MESP-2G of 0.99 US$/L.Scenarios H and K1 were modelled as Scenarios G and J respectively, but including leaves in the 2G production. This resulted in a net increase in ethanol production of 41 L/ton-dSC compared with the corresponding scenarios without leaves, obtaining a total of 136 L/tondSC of 2G ethanol. There is no streams integration between 1G and 2G ethanol production in Scenario H while Scenario K1 is fully integrated (heat and streams). In the latter case, integration allows a lower ethanol production cost resulting in a MESP-2G of 1.02 US$/L, compared to 1.05 US$/L in Scenario H. This is mainly due to the lower capital cost in Scenario K1. The enzymatic hydrolysis of the leaves is assumed to be performed at 14% WIS in Scenarios H and K1, which is twice that in Scenarios F and K2. Consequently, the sugar yield from EH falls by 20% due to the difficulty in hydrolysing the material at higher WIS, therefore leaving more EH solid residues for CHP production. For this reason, the electricity export opportunity cost decreases compared with Scenarios F and K2, but the decrease in net ethanol production, by 15 L/ton-dSC, causes an increase in MESP-2G. From an economic point of view it is better to perform the EH of leaves at 7% WIS than 14% WIS.Scenarios L1 and L2, shown in Figure The various scenarios simulated in this work give an indication of how a future process for the integration of 2G and 1G ethanol production from sugar cane could be designed in order to achieve the lowest 2G ethanol production cost. However, the number of ways in which heat and streams can be integrated is significantly larger than the number investigated in this project, and we are convinced that it is possible to further reduce the production cost in the future with other integration conditions.Table The calculated MESP-2G is very sensitive to the electricity selling price and to the cost of leaves and enzyme, thus an analysis of these variables was performed to investigate the impact on the MESP-2G in the most relevant scenarios. Figure The impact of enzyme cost on the MESP-2G in Scenarios D, F and L1 is shown in Figure The sugar cane accounts for a large proportion of the MESP of 1G ethanol production (61.3%). Nevertheless, changing this price has no effect on the MESP for 2G ethanol since it is calculated as a difference between the cost of 1G ethanol. Moreover, a change in the price of leaves would directly affect the MESP-2G, as can be seen in Figure In Figure Flowsheet models were developed for the design and simulation of various process layouts and for the assessment of the energy efficiency, capital and overall ethanol production costs in order to identify the process parameters and configurations having the lowest minimum ethanol selling price for 2G production.Without subsidies, the MESP-2G from bagasse and leaves (0.99-0.78 US$/L) can compete with starch-based ethanol from Europe. This is to some extent dependent on the method used to calculate the cost of the 2G ethanol, which can thus be significantly influenced by the opportunity costs and revenues acting on the 1G ethanol production. Indeed a high sugar price can contribute to increased 1G ethanol costs reducing the gap between 1G ethanol and 2G ethanol production costs. On the contrary, higher revenues from electricity export can yield a wider gap, decreasing the competitiveness of 2G ethanol. In fact, after subtracting the income gained from the electricity sale, the minimum selling price for 1G ethanol is reduced by 30%, from 0.38 to 0.26 US$/L.When the average ethanol selling price is 0.53 US$/L, the combined 1G + 2G ethanol production is economically feasible for all the cases simulated, even if the profitability is still lower (IRR < 21.5%) compared to 1G ethanol and electricity production (IRR = 32.1%). Higher yields in sugar recovery and conversion as well as a higher ethanol selling price might allow a possible profit breakeven of combined 1G + 2G ethanol with 1G ethanol.Drying the EH solid residues to 80% DM in a steam dryer before CHP production reduced the 2G ethanol production cost, as more heat and power were produced from the same amount of DM, resulting in more bagasse being available for ethanol production.It is important to obtain a high yield of fermentable sugars from the pretreatment and enzymatic hydrolysis steps. Doubling the enzyme dosage in the enzymatic hydrolysis can cause a lower MESP-2G, by about 12%, due to the increased ethanol production, of about 33%, as seen in Scenarios D vs. B. When leaves were used in addition to bagasse, the 2G ethanol production cost decreased in spite of the higher capital cost. The addition of leaves almost doubled the amount of 2G ethanol (compare Scenario F with Scenario D) as an effect of the higher sugar yield from leaves and the increase in electricity production due to more solids being available to the CHP plant. This resulted in a decrease in MESP-2G of almost 20%. However, when considering leaves, in the choice between "high WIS and lower yield in the EH" versus "low WIS and higher yield", the higher WIS pays off due to lower capital cost.It is also fundamental to decrease the energy demand in the process or to increase the electricity production in the CHP plant as the electricity price has a considerable impact on the 2G ethanol production, since less electricity is exported compared to the Reference scenario, i.e. 1G ethanol production in an autonomous distillery. The most preferable way of reducing the energy demand is by heat integration wherever possible. Another way is by using higher WIS concentration in EH because of the higher ethanol concentration and lower energy demand in distillation. An increase in electricity production in the CHP plant can be achieved by drying the solids before burning, as shown in Scenario B. Another interesting option is biogas production from the stillage streams, although calculations suggest that batch anaerobic digestion (AD) is not cost effective due to the considerably diluted streams. This can be improved in the future by continuous AD, but requires further studies.Finally, both the electricity price and the enzyme cost have considerable impact on the 2G ethanol production cost, as summarized in Table The overall methodology used for technical and economic assessment can be divided in three main steps: flowsheet design and simulations, capital cost evaluation and investment cost analysis.First, the flowsheets for the various process configurations were designed and simulated using the commercial software AspenPlus v7.1 (AspenTechnology Inc.) to perform rigorous thermodynamic calculations for mass and energy balances. Most of the process data implemented in AspenPlus were obtained experimentally within the project, such as bagasse and leaves composition , the recThe AspenPlus physical properties database contains most of the data needed for simulation of the entire process; but data for complex substances, for instance yeasts, enzymes and biomass components, such as lignin, were taken from the NREL physical properties database . For speThe output data from AspenPlus simulations were then used as input for the Aspen Process Economic Analyzer (AspenTechnology Inc.) for equipment sizing and for the estimates of both direct and indirect capital costs. For some specific equipment the cost was obtained from vendor quotations.® Excel spreadsheet starting from the capital cost and the operating costs. The profitability of the various process configurations investigated was expressed as the minimum ethanol selling price for the 2G (cellulosic) ethanol (MESP-2G).Finally, the overall ethanol production cost was calculated in a MicrosoftSecond-generation ethanol production from sugar cane residues can be positively influenced by integration with the 1G ethanol process and the benefits obtained depend on the level of process integration. Moreover, the operating conditions for the 2G process can also improve the overall economic feasibility. The aim of this study was to compare different operating conditions, process options and levels of integration for ethanol production from whole sugar cane in terms of energy efficiency and capital cost, in order to identify the lowest MESP.3PO4-catalysed steam-pretreated bagasse and leaves. The CHP plant provides heat and power either to the 1G plant or to the combined 1G and 2G processes by burning bagasse, EH residues and biogas produced by AD.A full-scale plant was modelled and the following four main processes were simulated: 1G ethanol production from sugar cane juice, 2G ethanol production from lignocellulosic residues of the sugar cane (bagasse and leaves), combined heat and power (CHP) plant and waste water treatment by anaerobic digestion (AD). The 1G process (autonomous distillery) comprises the traditional Melle-Boinot steps, well-experienced so far in the sugar cane-to-ethanol industry, while the 2G ethanol production is based on SHF of HBesides the process configurations, process variables found to have significant impact on the plant design and capital cost, based on experimental results obtained within the project, were investigated. These were mainly parameters associated with the enzymatic hydrolysis step, i.e. the residence time, enzyme dosage, concentration of water-insoluble solid material (WIS) in the pretreated material, as well as the addition of sugar cane leaves to raw material for 2G ethanol production. Among the pretreatment conditions tested experimentally for bagasse and leaves in the project, those resulting in the best EH yield were selected for model simulation.The autonomous distillery modelled as it is today was used to represent the actual state of the art of an ethanol distillery using sugar cane juice in Brazil 1G), and was also considered as the initial flowsheet to which the 2G process was added  and two rectifiers (56 trays each) arranged in parallel.Sugar cane shredding and milling are the first processing stages, in which the juice used for fermentation in the 1G plant is separated from the bagasse, which is combusted in the CHP plant. The latter is used for the production of steam and electricity allowing the mill to be self-sufficient in energy, and providing excess electricity that can be sold. The sugar cane juice is purified by adding CaO and a flocculant polymer, followed by clarification. Since the sucrose concentration is too low (13.7 wt-%) to reach the desired ethanol titer in the fermentation stage, an evaporation unit is used to increase the concentration to 19 wt-%. The unit is composed of two evaporators in parallel, each with an area of 3500 mel) and steam to the whole plant. The isentropic efficiency of the turbines was assumed to be 0.80. The steam for the plant is withdrawn at 144°C and 2.5 bar. In the autonomous distillery the material used in the CHP plant is the sugar cane bagasse at a dry matter (DM) content of 50% from the mill section. When bagasse is used in the 2G ethanol production process, the material combusted in the CHP plant consists predominantly of the solid residues after EH, mainly lignin, with the addition of some bagasse when the solid residues are not sufficient to meet the demand for steam from the 1G and 2G processes. When leaves are also used for ethanol production the material used in the CHP plant consists only of the solid residues after EH of bagasse and leaves. In the simulated scenarios, the boiler size was based on the mass flow feed rate of solids to the boiler, and the turbine system was modified to the steam requirement as the 2G process. Several turbine withdrawals were designed to supply superheated 20 bar steam to the dryer, saturated 10 bar steam for pretreatment, and 2.5 bar steam for evaporation and preheating of the feed to EH and distillation. A condensing turbine operating at 0.11 bar was also added when the steam requirement for the plant was lower than the total amount of steam produced.Figure Figure 3PO4  and then preheated to 95°C by direct injection of low-pressure secondary steam, before being fed to the continuous steam pretreatment reactor. Due to the high flow rate of feedstock two pretreatment reactors are needed for the bagasse. In the scenarios where leaves are also used a third pretreatment reactor is needed. The temperature in the reactor is maintained at 180°C by injecting high-pressure saturated steam at 10 bar, and the residence time is 10 minutes for both bagasse and leaves. Heat losses are assumed to be 10% of the adiabatic heat demand. The pretreated material is then flashed in three pressure reduction steps , and the flash vapours obtained are condensed to heat other streams in the plant. After flashing part of the water in the slurry, the WIS concentration is 16%. The pretreatment step is the same in all scenarios.Bagasse (and leaves when added) are impregnated with H3 and washed in a filter press to recover the solubilized sugars, mainly pentoses, in the liquid fraction. The filter cake of solids content (35 wt-% DM) is diluted to obtain the desired WIS content, and enzymes are added for the EH process, which is performed at a temperature of 50°C. To investigate the effect of integrating the 1G and 2G processes, various residence times, enzyme loadings and WIS concentrations in the EH step were used. Yields of released glucose as a function of the enzyme dosage and residence time were based on experimental data, see Table 3, resulting in 32 to 70 tanks. After EH, a filter press is used to separate the fermentable broth from the partially hydrolysed solid residues, which are sent to the CHP plant.The slurry from the last flash step in the pretreatment is neutralized to pH 5 with NH3, resulting in between 8 and 12 fermentors. The yeast is recovered by centrifugation prior to the liquid fraction entering the distillation unit, and low-pH washing of the yeast prevents any possible infections. The ethanol from the fermentation step is concentrated by distillation to 92.8 wt-%. The distillation section consists of two stripper columns (24 trays each), and a rectifier (36 trays), which are heat-integrated by operating at different pressures. The feed from the fermentation stage is divided between the two strippers. The first stripper is heated with live steam while the second stripper is heated by the overhead vapour from the first stripper. The rectifier is heated by the overhead vapour from the second stripper. Ethanol recovery was assumed to be 99.5% in each column.The liquid is then fermented using an industrial yeast strain from CTC, giving 94% of the theoretical ethanol yield from hexose sugars. The complete cycle, which includes filling, fermentation, draining and cleaning, lasts 8.2 hours, and the size of the fermentors was set to 1200 mThe integrated 1G and 2G plant has many waste streams with a high COD content that can be treated by AD to produce biogas, which is burnt in the boiler for steam and electricity production. These streams include the bottom streams from distillation , the condensates obtained during pretreatment, evaporation and drying, and the pentose-rich liquid fraction from filtration of the pretreated material prior to enzymatic hydrolysis. Experimental trials were performed to obtain data for the calculation of the biogas potential from these streams. These data are required as input to the flowsheet calculations and are fundamental in the energy balance of the integrated 1G and 2G system, since the amount of biogas can be important in improving the energy supply and reducing the final ethanol production cost. The biogas potential was measured in batch trials for several stillage streams and for the pentose-rich stream after filtration of the pretreated material (data not shown).CH4/gCOD for the stillage and pentose-rich streams from bagasse, respectively. Table 3 each would be required, and the investment cost for the biogas facility alone would be about 68 million US$. For this reason, only AD of the pentose-rich stream, which has a higher content of COD and requires 98 hours residence time to reach 96% of the maximum biogas production with a yield of 0.116 gCH4/gCOD, was included in the final simulations.The microorganism consortium was taken from the Domsjö Fabriker AB spent sulphite liquor plant . The stillage streams required at least 10 days residence time in AD, whereas the pentose-rich samples reached maximum production after about 5 days. Continuous AD could probably shorten the residence time, but the results for the batch system were used to be on the conservative side. The final methane yield was 0.112 and 0.127 gWhen the 2G plant is simply annexed to the existing 1G facility, there is no integration except the sharing of heat and power produced in the CHP plant. Thus the synergies arising from the combination of the two processes cannot be exploited to minimize the energy demand and the capital cost. Alternative configurations for the combined 1G + 2G facility rely on the strategy adopted for integrating the 1G and 2G processes in a new plant. For this reason, energy reduction and recovery, as well as process stream mixing are the integration options that were investigated to find the configuration with the lowest MESP-2G. Energy integration is achieved by further increasing the use of new and more efficient equipment, suitable for recovery and/or in a different configuration. However, new equipment can significantly increase the capital investment cost, thus the economic feasibility of better energy use was also evaluated. Process energy efficiency was defined as the energy output in the products (ethanol and excess electricity) divided by the energy input, i.e. the energy contained in the raw materials . Calculations were based on the HHVs of the raw materials and ethanol. The electric power was recalculated in terms of the fuel necessary to produce this electricity, assuming an electricity-to-fuel ratio of 0.33.Regarding stream integration, mixing of material flows can have beneficial effects on the energy balance, especially in the distillation and evaporation stages. Two mixing options were considered in an attempt to reduce the energy demand in these two steps: mixing of the 1G ethanol-rich stream and the 2G ethanol-poor stream prior to the distillation unit . The fixed capital investment cost was recalculated from 2008 to 2010 using cost indices for Houston, Texas, USA. A factor of 0.82 was then applied to scale fixed capital investment costs from the US gulf coast to Brazilian conditions. These data, together with stream data, were exported from Aspen Process Economic Analyzer and used in the Excel model developed for the final cost calculations.The results from AspenPlus simulations  were imported into Aspen Process Economic Analyzer v.7.1  where equipment was sized and direct/indirect costs were determined. Fixed capital investment costs were estimated based on costs for the first quarter of 2008, except for the cost of the pretreatment unit, which was obtained from a vendor quotation based on pretreatment using SOThe Excel model comprises investment costs and data on biomass, chemicals, enzymes, electricity price and all other running costs. The main premises for the production cost estimate are that it is a stand-alone ethanol production plant processing 165 dry metric ton of sugar cane per hour and is operated 200 days per year. The plant is equipped with a CHP plant for production from residue and/or sugar cane leaves. Excess electricity is exported.th plant, meaning that it is considered to be based on known technology at the time of construction (N could be 3-6 depending on the size and complexity of the demonstration plant). The Minimum Ethanol Selling Prices (MESPs) for the integrated 1G + 2G process and the separate processes are calculated as the sum of each single production cost item  and positive for the 2G plant . The plant is assumed to be the Ni 2G Pis the 2G cellulosic ethanol production cost for the item i given by a weighted ratio between the difference in the cost of item i for 1G + 2G and 1G ethanol and the volume of 2G ethanol produced. The data for 1G ethanol production, indicated by the index 1G, were derived for the autonomous distillery (Reference scenario). i Pdenotes the production cost for item i and F the amount of ethanol produced in the specific process. The MESP is not only a measure of total production cost but also includes the investment return, accounted for as capital cost, at the desired internal rate of return (IRR). The MESP was varied in the spreadsheet model until the net present value (NPV) equalled to zero at the selected IRR (10%). The NPV was calculated based on the values given in Table where # European Commission-funded project within the 7th Framework Programme in partnership with Brazil. Period March 2009 - February 2011. Project participants: Centro de Tecnologia Canavieira, Piracicaba, SP, Brazil; Universidade Federal do Paranà, PR, Brazil; Novozymes Latin America, Brazil; Novozymes Denmark; and Lund University, Sweden.1G: first generation; 2G: second generation; 1G + 2G: combined first and second generation; AD: anaerobic digestion; CHP: combined heat and power; DM: dry matter; dSC: dry sugar cane; EH: enzymatic hydrolysis; MESP: minimum ethanol selling price; SC: sugar cane; SHF: separate hydrolysis and fermentation; SSF: saccharification and simultaneous fermentation; WIS: water insoluble solidsThe authors declare that they have no competing interests.SM carried out the simulations and economic evaluation and wrote the paper. JM developed the model for financial calculations. GZ, JM and SM designed and analysed the study. GZ and JM reviewed the paper. All authors read and approved the final manuscript.


Rhammatocerus brasiliensis.Supernumerary B chromosomes occur in addition to standard karyotype and have been described in about 15% of eukaryotes, being the repetitive DNAs the major component of these chromosomes, including in some cases the presence of multigene families. To advance in the understanding of chromosomal organization of multigene families and B chromosome structure and evolution, the distribution of rRNA and H3 histone genes were analyzed in the standard karyotype and B chromosome of three populations of the grasshopper R. brasiliensis it was also observed multiple sites for H3 histone genes, being located in the same chromosomal regions of 5S rDNAs, including the presence of the H3 gene in the B chromosome.The location of major rDNA was coincident with the previous analysis for this species. On the other hand, the 5S rDNA mapped in almost all chromosomes of the standard complement (except in the pair 11) and in the B chromosome, showing a distinct result from other populations previously analyzed. Besides the spreading of 5S rDNA in the genome of R. brasiliensis, their chromosomal distribution was not informative in the clarification of the origin of B elements. Our results indicate a linked organization for the 5S rRNA and H3 histone multigene families investigated in R. brasiliensis, reinforcing previous data concerning the association of both genes in some insect groups. The present findings contribute to understanding the organization/evolution of multigene families in the insect genomes.Due to the intense spreading of 5S rRNA and H3 histone genes in the genome of  In eukaryotes the transcribing sequences for ribosomal RNAs (rRNAs) are organized in two multigenic families tandemly arrayed in the genomes. The major one (45S rDNA) transcribes the 18S, 5.8S and 28S rRNAs and the minor one (5S rDNA) is responsible for the transcription of the 5S rRNA .Due to the clustered organization of multigene families and other repetitive DNAs they have been an important source as chromosomal marker for analysis of karyotypic evolution, genomic structure and origin and evolution of B chromosomes in animals. In grasshoppers the mapping of repeated elements is primarily concentrated in analysis of number and location of rDNAs and histone genes, and in the lesser extend satellite DNA (satDNAs) -9. ConceEyprepocnemis plorans and Locusta migratoria. In this group variability for B chromosomes related to heterochromatin patterns, satellite DNAs organization and multigene families has been reported .All populations of o et al. ,18. A toin situ hybridization of the 18S rDNA probe revealed that this sequence was located in the pericentromeric region of three autosomal chromosomes  in all populations analyzed (Figure The d Figure . The 5S d Figure . The duad Figure .The H3 histone gene was only observed in the pair M7 in one individual from Surubim Figure , while fR. brasiliensis is coincident with the results published by Loreto et al. [R. brasiliensis the most plausible explanation is the transposition or occurrence of ectopic-recombination of major rDNA sites, since this species maintains the ancestral diploid number for Acrididae, and no evident macro-chromosomal rearrangement was observed in its karyotype. On the other hand, for other Gomphocerinae the action of large chromosomal rearrangements were important in changing of rDNA location, as described for species with 2n = 17,X0 [The number of sites for 18S rDNA observed in the three populations of o et al. ,18 for to et al.  the vario et al. ,20. For  = 17,X0 .R. brasiliensis besides the spreading of major rDNA sites it was also observed a more intense dispersion for the 5S rDNA, for almost all chromosomes of the complement. High variability for 5S rDNA cluster number was also recently observed for other Acrididae species [Allium [The mapping of 5S rDNA in grasshoppers was performed in about 34 species, including four Proscopiidae and some Acrididae, being the number and chromosomal location conserved in the former and variable in the later -10. In R species . The abi [Allium , and rec [Allium -24.R. brasiliensis, being the ancestral characteristic the occurrence of only one site that occasionally was observed in the population sampled from Surubim. The spreading of H3 histone can be directly associated to the dispersion of 5S rDNA, indicating a linked organization of these two multigene families in the genome of R. brasiliensis, which causes the same evolutionary patterns for the two sequences. On the contrary, the analysis of other 29 Acrididae species revealed a distinct scenario, mainly without association of 5S rDNA/H3 histone sequences, being these elements co-located only in five species, but with non co-dispersion in species with 5S rDNA spreading (see Table R. brasiliensis and in other insects to confirm the genomic nature of the association of 5S rRNA and histone H3 genes.FISH using H3 histone sequence as probe also revealed dispersion for this gene sequence, being the pattern coincident to the same location of 5S rDNA, confirmed by two color FISH. In 35 species of Acrididae and four species belonging to Proscopiidae grasshoppers the number and location of histone genes were extremely conserved, with the presence of only one site ,8. CabreR. brasiliensis, being identical the position of 18S rDNA observed here. Considering that the location of 18S rDNA is stable among the populations of R. brasiliensis, the co-location of ribosomal elements in the three studied populations might be explained by invasion of 18S rDNA or a near region by the 5S rRNA genes through transposition events. The movement of 5S rDNA through transposition has been documented for example in mammals [R. brasiliensis there is no evidence of interspersion of 18S rDNA and H3 histones genes, considering that the distribution in the histone gene clusters is not followed by dispersion of 18S genes.Besides the co-localization of 5S rDNA and H3 histone sequences it was also observed a similar pattern for 18S and 5S rDNA in the chromosomal pairs M4, M6 and S9 of all populations. Loreto et al.  describe mammals  and fish mammals . Associa mammals -31. On tR. brasiliensis is widely distributed among all population analyzed until the present time. Moreover a possible autosomal origin for this element using in situ hybridization for 5S and 45S rDNAs was proposed [R. brasiliensis due the presence of 5S rDNA sites in almost all autosomes and also in the X chromosome, being this chromosomal marker non informative for B origin in this species. An alternative explanation can be the multiple origin of the B element in the distinct populations of R. brasisiensis studied with distinct conditions of 5S rDNA location. Multiregional origin for B chromosomes in grasshoppers was also previously proposed by Cabrero et al. [E. plorans, using the mapping of rDNAs and a satellite DNA with 180 bp repeat sequences. Another limitation of the use of 5S rDNA sequence as an informative marker for definition of B origin and evolution is related to the intense evolutionary dynamics of this sequence in grasshopper genomes, as described by Cabral-de-Mello et al. [R. brasiliensis could be a characteristic generated by transposition after the origin of this chromosome, and not a characteristic inherited from the chromosome responsible for its origin.The B chromosome of proposed . This coE. plorans and in some other animal groups, such as fish and mammals [L. migratoria, being a good marker for discrimination of ancestry of this chromosome in this species [R. brasiliensis is the second case for grasshopper and for animal as a whole, indicating that in fact these chromosomes can harbor more gene sequences than was observed until now. Moreover, due the clustered organization of histone genes they can be a useful marker in the investigating of B origin. On the other hand, in the case of R. brasiliensis, this sequence, as well as the 5S rDNA, was not informative due their high dispersion. The presence of H3 gene in the B chromosome can be also directly associated with the dispersion of 5S rDNA, reinforcing the co-dispersion/co-localization of these two multigene families.Besides the presence of 5S rDNA in B chromosomes of grasshoppers, satellite DNAs and 45S rDNA were also identified in this genomic element, as for example, in  mammals -34. On t species . The preR. brasiliensis the ribosomal DNAs and histone genes apparently are not sufficient for definition of B origin, due their intense evolutionary dynamics leading to spread of clusters in the genome and remarkable differences among population of the species.The results presented here reinforce the hypothesis of co-localization/association of the 5S rDNA and H3 histone genes in some insect groups, and other invertebrates. This association led to the same patterns of chromosomal dispersion of these two multigene families in the genome, including both A complement and B chromosomes. These results also contribute to a better knowledge about B chromosomes content in animal kingdom. Moreover, we bring attention in the definition of chromosomal markers to be applied for analysis of B origin, depending on the mechanisms of evolution for the chosen sequence in the genomes to be investigated. Specifically for the case of The authors declare that they have no competing interests.NLO and DCCM carried out the conventional and molecular cytogenetic analysis and draft the manuscript. VL and MFR helped in the discussion of the obtained data. RCM, CM and DCCM conceived of the work, and participated in its design, drafted and revised the manuscript. All authors analyzed all results and read and approved the final manuscript. DCCM revised the manuscript with the suggestions from the reviewers.


Therefore the inviduals with this disorder can be reared as boys or girls. Most of Indonesian DSD cases are facing with budget constraint, because they require series of costly examinations and treatment.We aimed to know the distribution of sex rearing in inviduals with 46,XY DSD and tried to find out the possible etiologies based on the clinical manifestations and the age of presentation.The study reported data of types of sex rearing, age at and clinical presentation of subjects obtained from 46,XY resulted chromosome analysis patients referred to our clinic in year 2009-2010.Seventy 46,XY DSD cases (aged 1 day-32 years old) were collected and analyzed, and 45 of them were reared as boys, 22 as girls, while the types of sex rearing was not yet assigned in 3 cases. Most of the cases were referred for chromosome analysis at age > 3 months-8 years, and 12 patients at 18 years or above, 10 of them were raised as female. Genitalia ambiguity was the most frequent referral reason (44/70 cases). Based on detailed clinical examinations and age of presentation we also described the possible diagnosis, in order to help parents or patients to assign gender with minimum cost.


According to many authors, acute necrotizing pancreatitis (ANP) still remains one of the difficult problems of abdominal surgery. The complexity of the pathogenesis of the disease, features of the pancreas pathomorphology, abdominal hypertension, and high mortality (30 to 70%) necessitate a search for new ways to treat this disease.n = 23) had epidural analgesia by ropivacaine 6 to 14 mg/hour during 7 to 10 days, and from the second group (n = 21) opioid analgesia by trimeperidine 20 mg three times a day during the same period. We monitored the level of septic and thrombohemorrhagic complications by clinical and instrumental data, during the month after treatment starting. The hemostatic system was evaluated using indicators of hemoviscoelastography .The study was conducted in 44 patients with ANP, who were divided into two groups according to type of analgesia: epidural or opioids. Patients from the first group  to 8.7% (first group). We think that violations of blood coagulation and microcirculation are the basis for ischemia, necrosis in tissues and septic complications. Epidural analgesia is an effective method to decrease the level of septic and thrombohemorrhagic complications and mortality in ANP patients.


The human gut microbiota play a vital role in health and nutrition but are greatly modified during severe diarrhoea due to purging and pathogenic colonization. To understand the extent of loss during and after diarrhoea, faecal samples collected from children (n=21) suffering from acute diarrhoea and from their healthy siblings (n=9) were analyzed by 16S rRNA gene-targeted universal primer polymerase chain reaction (PCR), followed by temporal temperature gradient gel electrophoresis (TTGE). The gut microbiota decreased significantly as indicated by the number of TTGE bands at day 0 of acute diarrhoea . The number of bands showed a steady increase from day 1 to day 7; however, it remained significantly less than that in healthy siblings . These results suggest that appropriate therapeutic and post-diarrhoeal nutritional intervention might be beneficial for the early microbial restoration and recovery. Faecal samples were collected from 21 children at day 0 (after enrollment and before any medication), at day 1 (after 24 hours), day 2, day 3, and day 7. As a standard management, they received glucose-based oral rehydration solution for rehydration and also received the usual antibiotic therapy, i.e. a parenteral ampicillin dose of 100 mg/kg in four to two equally-divided doses for five days. Faecal samples were also collected once from nine non-diarrhoeal children aged 12-18 months (median 12 months) from siblings of the patients to compare with diarrhoeal children. These children resided at their homes, did not present with diarrhoea, and did not take antibiotics during the last two months.et al.  and reverse primer S-D-Bact-788-a-A-19 (5’ GGA CTA CCA GGG TAT CTA A 3’) were used for amplifying the variable region 3 and 4 of the bacterial 16S rRNA genes in PCR . A GC-riof 20 µL . PCR ampm longum . Gels weet al. who developed the original strategy . The previously-described analysis is based on the method of Deplancke strategy . Comparistrategy . The Dsct-test was used for comparing the significant differences between the mean values.Data for the band numbers and distances for different time periods were calculated as mean±standard error of mean, and Student's A PCR-TTGE analysis of dominant bacteria from the 16S rRNA gene of five different faecal samples from each patient revealed various profiles comprising 3-23 bands. At day 0, the number of bands in 14% (3/21) of the children was between 3 and 5; it was between 6 and 9 in 29% (6/21) of the children; and it was between 12 and 15 in the remaining 57% (12/21) of the children. Before any treatment, the average number of bands at day 0 was 10.7±0.9 (mean±standard error) . CompareThe TTGE band profile of each patient was analyzed using Dice similarity coefficient to examine the re-establishment of the microbiota in the gut ecosystem. First, the distance of the band pattern was calculated from the first sample collected between day 0 and day 1, between day 0 and day 2, and so on. Second, the distance between two consecutive samples was calculated between day 0 and day 1, day 1 and day 2, and so on. The mean values of these distances were analyzed to obtain a general picture of the faecal microbiota re-colonization in children with diarrhoea. In the first set of analyses, the TTGE profiles of the patients with acute diarrhoea were compared with the recovering stages , and the average distances were calculated representing the succession of faecal microbiota over these time periods . In the The study investigated the possible effects of diarrhoea on the diversity of the gut microbiota in children with acute diarrhoea, using a culture-independent metagenomic tool—TTGE. The results presented herein demonstrate a transient reduction in the bacterial diversity that took place after the onset of acute diarrhoea in children. This concurs with results published in recent culture-based studies in which children with diarrhoea were shown to reduce the number of bacterial species ,7,23. ThIn the present study, children suffering from diarrhoea received an appropriate rehydration therapy together with antimicrobial therapy and continued to be fed as per the guidelines of the World Health Organization. Although antimicrobials exert a beneficial effect by eliminating pathogenic agents, thereby controlling infections, the oral administration of antimicrobial agents often causes a shift in the intestinal microbial populations, with a decrease in anaerobic bacteria and a concomitant increase in aerobic bacteria ,26. In tE. coli in the reference lane) reflecting selective proliferation of a particular species was observed during day 1-3, the overall TTGE profiles for the antibiotic therapy period were more or less static, except that the banding positions were not consistent for every patient that we tested. By contrast, this pattern changed again when the antibiotic therapy ended, and a significant bacterial diversity in terms of numbers and positioning of the dominant bands was observed at day 7. One possible reason for this observation could be that fewer bacterial species could have proliferated in the presence of antibiotics during the clinical period when the sensitive population must have been eliminated/suppressed. This supposition is supported by results of a recent culture-based study in which administration of ampicillin to humans was shown to induce β-lactamase production by various intestinal microorganisms, including Enterobacteriaceae and some members of the Bacteroides fragilis group, which allowed simultaneous multiplication of some otherwise ampicillin-sensitive bacteria, such as Clostridium difficile  was enumerated and considered the baseline, and the changes occurring over time in the following days were taken into account as variation. Although a dominant band (bands parallel to ifficile ,28.In the present study, the TTGE profiles of diarrhoeal and non-diarrhoeal children varied greatly in terms of species richness as revealed by the number of different TTGE bands representing different species. These differences in the numbers of bands were in agreement with the values published in an earlier study  which anThis study has limitations as the number of samples was low. Another limitation was the sampling timeframe because one week was not enough for the displaced microbiota to be restored in the gut of post-diarrhoeal children. We believe that follow-up of the patients for an extended period of time would be needed to get a clearer picture of status of the gut microbiota in post-diarrhoeal children. Although the identification of TTGE bands representing different species would be interesting, in the present study, the intensity of the majority of the bands was very low and was not suitable for the purpose of purification of DNA by cutting TTGE bands for sequencing and analyses. Again, the positions of bands varied greatly in day 1 to day 3; so, extensive cloning and sequencing of dominant bands during treatment were also not suitable.The results of this study clearly showed that the bacterial diversity of the gut microbiota was significantly reduced during acute diarrhoea and that the diversity did not reach the level of non-diarrhoeal control children after one week. It is presumed that nutritional and appropriate therapeutic intervention during the post-diarrhoeal phase might be beneficial for achieving a higher magnitude of the diversity of the gut microbiota similar to that found in non-diarrhoeal children in a shorter duration.The study was conducted at icddr,b with support from Core Fund. The authors acknowledge the excellent service and assistance of the physicians and nurses at the Dhaka Hospital of icddr,b.


Debates have focused on population transitions between the bearers of the Middle Palaeolithic Aterian industry and the later Upper Palaeolithic populations of the Maghreb, as well as between the late Pleistocene and Holocene.The archaeology of North Africa remains enigmatic, with questions of population continuity Improved resolution of the mitochondrial DNA (mtDNA) haplogroup U6 phylogeny, by the screening of 39 new complete sequences, has enabled us to infer a signal of moderate population expansion using Bayesian coalescent methods. To ascertain the time for this expansion, we applied both a mutation rate accounting for purifying selection and one with an internal calibration based on four approximate archaeological dates: the settlement of the Canary Islands, the settlement of Sardinia and its internal population re-expansion, and the split between haplogroups U5 and U6 around the time of the first modern human settlement of the Near East.A Bayesian skyline plot placed the main expansion in the time frame of the Late Pleistocene, around 20 ka, and spatial smoothing techniques suggested that the most probable geographic region for this demographic event was to the west of North Africa. A comparison with U6's European sister clade, U5, revealed a stronger population expansion at around this time in Europe. Also in contrast with U5, a weak signal of a recent population expansion in the last 5,000 years was observed in North Africa, pointing to a moderate impact of the late Neolithic on the local population size of the southern Mediterranean coast. Key issues concern the identity of the bearers of the Middle Palaeolithic (or Middle Stone Age) Aterian industry  to ≥115-40 ka [Nassarius shell beads, use of ochre and bone tools, and long-distance exchange networks - preceding those of southern Africa and making it likely that the Aterian was carried by anatomically modern (rather than archaic) humans [Despite much recent research, the archaeology of North Africa remains enigmatic, with questions of population continuity ) humans . The fatversus replacement surround discussion of the widespread Epi-Palaeolithic Capsian industries, which saw expansion southwards from eastern Algeria into an increasingly humid Sahara in the early Holocene and sometimes associated with more gracile Mediterranean skeletal remains, and the subsequent emergence of the Neolithic [Further debates have focused on the question of population replacement in the late Pleistocene and Holocene. The earliest Upper Palaeolithic industry in North Africa is the Dabban, limited to Cyrenaïca  and moseolithic ,7-10.en route or within North Africa  has the potential to address questions such as these . In partn Europe ), the prn Europe .These early studies were based only on information from hypervariable region I (HV-I) in the mtDNA control region and a small number of diagnostic RFLPs in the coding region, assayed in samples from several populations of North Africa and Iberia. In most of these regions, U6 frequencies are ~10% or less , and eveBy contrast, U6b (characterized by variants at 16172-16219-16311) has a more limited range to the northwest of North Africa, the north of the Iberian Peninsula and, as a nested derivative , in the Canary Islands . In parteastwards in Palaeolithic times, and U6a1 a further reverse movement from East Africa back to the Maghreb, possibly coinciding with the probable Afroasiatic linguistic expansion. The clades U6b and U6c, restricted to West Africa, had more localized expansions; they argued that U6b reached Iberia at the time of the diffusion of the Capsian culture in North Africa.Maca-Meyer et al.  performevia the Levant, possibly during the Greenland Interstadial 12, from ~44-48 ka. They reaffirmed that the various U6 sub-groups originated in the southern Mediterranean area, dispersing subsequently to East Africa.However, a larger study by Olivieri et al.  was closin situ rather than prior to the presumed founder event, the estimate of the time to the most recent common ancestor (TMRCA) can provide a minimum bound on the age of the migration event . Howevenalysis" . ConsideWith respect to the molecular clock, there are many factors leading to uncertainty. There is the wide variation in positional mutation rates and violations of the independence of mutations at different positions. Obvious regions are the paired stems of rRNAs and tRNAs , which sHomo sapiens, rendering the application of a strict clock problematic [The choice of calibration points is also an important issue. Traditionally, an outgroup is used, where the split time with the human lineage can be assigned in some way. For humans, the closest one is that corresponding to the human-chimpanzee split, for which the fossil evidence is controversial and which is in a time frame very distant from the TMRCA of the mtDNA of blematic . One recblematic . A stratblematic  is appeablematic  advocateblematic  applied blematic , and resblematic ), based blematic .Here, we analyse an additional 39 complete U6 genomes, from the full range of the U6 geographic distribution, including the Near East, Iberia and the Canary Islands. This has enabled us to construct a phylogenetic tree including 89 U6 genomes in total, and to re-evaluate the demographic history of the haplogroup, and its role in North African prehistory, in the light of the recent developments in the calibration of mutation rates. A comparison tree was inferred for 141 U5 sequences from the literature, allowing us to test the use of four alternative internal calibration points  against the recently developed complete genome clock with a correction for purifying selection.th WMA General Assembly, Seoul, October 2008) and was approved by IPATIMUP Ethics Commission.Based on information from HV-I that allowed the general classification into haplogroups, we selected 39 U6 individuals for complete sequencing: 22 from Portugal, most of them included in a published Portuguese database  and someHQ651676-HQ651714).We amplified mtDNA using 32 overlapping fragments as described elsewhere . After pFor the U6 phylogeny reconstruction, besides the new 39 sequences obtained here, we used 50 complete sequences previously published ,40,44-47th percentile were assigned.For estimation of the TMRCA for specific clades in the phylogeny, we used ρ statistic frequentist, maximum likelihood and Bayesian phylogenetic analyses. We used the ρ statistic  with a mutation rate estimate for the complete mtDNA sequence of one transition in every 3,624 years , correctth percentile of 60 ka for the split between the U5 and U6 lineages, based on the hypothesis that some of the first settlers of Europe [- For the first point, we assigned a mode age of 45 ka and a 95f Europe  were carf Europe . Consideth percentile 5 ka was the age of the U6b1 branch based on the archaeological date for the colonization of the Canary Islands. The first settlement of the Canary Islands is extremely uncertain and the archaeology of the islands is not extensive [- The second calibration point with mode 2.3 ka and 95xtensive . Furtherth percentile 14 ka was within U5b3a1, a subclade of which, U5b3a1a, is found only on Sardinia [- The third point with mode 8 ka and 95Sardinia . SardiniSardinia . The colSardinia . Again t- The fourth calibration point we used was an age estimate for the Sardinian U5b3a1a branch, assigned a normal distribution (truncated at zero) centred on 5.8 ka, with a standard deviation of 1,000 years. After the settlement of Sardinia, the population size was probably low (considering the long branch between U5b3a1 and the ancestor of U5b3a1a). It is tempting to identify the subsequent rather star-like radiation of U5b3a1a with a population expansion. The late Neolithic in Sardinia around 5-6 ka was a likely time of internal re-expansion and population increase  and we hBEAST uses a Markov-chain Monte-Carlo (MCMC) approach to sample from the posterior distributions of model parameters (branching times in the tree and substitution rates). Specifically, we ran 150,000,000 iterations, with samples drawn every 1,000 MCMC steps, after a discarded burn-in of 15,000,000 steps. We checked for convergence to the stationary distribution and sufficient sampling by inspection of posterior samples. We also obtained Bayesian skyline plots from BEAST and visualised them with Tracer v1.3 from posterior distributions of parameters run for 50,000,000 iterations . We used a generation time of 25 years to rescale the vertical axis of the BSP to years. In addition, we forced the larger sub-haplogroups  to be monophyletic in the analysis, as the presence of fast-evolving positions (such as 16189 and 16311) leads to the reconstruction of diverse phylogenies, which would not be comparable with the one inferred using network analysis; MCMC updates which violated this assumption were immediately rejected.http://www.esri.com/software/arcview/. We used the "Inverse Distance Weighted" (IDW) option with a power of two for the interpolation of the surface. IDW assumes that each input point has a local influence that decreases with distance. The geographic location used is the centre of the distribution area from which the individual samples of each population were collected. The data used are listed in Additional File To determine and visualise the geographical distribution of U6, U6a and U6bd, we constructed interpolation maps using the "Spatial Analyst Extension" of ArcView version 3.2 The enlarged U6 phylogeny  existing archaeological evidence indicates  or that It is also worth pointing out that the age of haplogroup U reported here (the U5-U6 split at around 46 ka) is slightly lower than the age of U reported recently using ρ and ML and the complete sequence clock corrected for purifying selection ,31 whichA further check of the clock is the age of U5a2a , since aThe BSPs Figure  computedThe BSP for U5 shows a small effective population size from the time of its origin until a strong expansion led to a ~11-fold increase within a period of ~6 ka in the late Pleistocene/early Holocene (14.3-8.3 ka). Following this, the effective size again remained more or less constant till a second strong expansion occurred, leading to an increase of ~5-fold after 4.3 ka (we took the year 1600AD as the most recent time-point ). For U6The Bayesian evolutionary analyses conducted in this work, as well as the comparison between recently updated mutation rates and the improvement of the phylogenetic resolution for the mitochondrial haplogroup U6, enable us to revise our interpretation of the evolutionary history of haplogroup U6 and sketch a demographic prehistory for North Africa, which we can then compare with that of Europe. In particular, they allow us to address many of the archaeological questions raised in the introduction.The recently revised archaeological dates for the Aterian industry of North Africa emphasize that the makers of this industry do not appear to have left any imprint in the maternal lineages of present-day North Africans. The oldest arrivals amongst extant mtDNAs appear to be the U6 and M1 lineages, which date to 36.6  and 25.4  ka respectively . As withThere is an intriguing further signal in the U6 data, witnessed by the Bayesian skyline plot. For the European haplogroup U5, which is one of the most ancient in Europe , we idenWe can compare the U5 and U6 BSPs with the ones for geographic regions published in Atkinson et al. , inferreThis observation suggests that by investigating in depth U6 and U5, the oldest lineages present in North Africa and Europe, respectively, we are indeed receiving signals from the demographic pre-history of modern humans in these regions.Aside from U6, North Africa was also the recipient of European, Near Eastern and sub-Saharan African lineages most of which most likely arrived in the Holocene. Haplogroups H1, H3 and V expanded in Iberia in the Lateglacial/postglacial ,58-61, aMost sub-Saharan lineages observed in North Africa are presently difficult to date and probably arrived at various times, but the age of the sub-Saharan subclade L3e5 indicates its arrival in North Africa from the south ~7 ka, following its expansion in the immediate postglacial humid phase ~11.5 ka . Other LLP conceived the study, coordinated its design and drafted the manuscript. RFD, VF, JBP and MDC carried out the molecular genetic studies, participated in the sequence alignment and in the phylogeny reconstruction. MBR and HM revised the manuscript, in particular focusing on the archaeology of North Africa and interpretation of the genetic results against the archaeological background. NMS and PS performed the statistical analyses and interpreted results with LP, DMB, MBR and VM. All authors read and approved the final manuscript.List of complete U6 and U5 samples and HV-I samples. List of complete U6 and U5 samples used in this work, and of HV-I samples for the spatial smoothing analyses.Click here for filePhylogeny of the complete U6 mtDNA sequences. Phylogenetic tree reconstruction for 89 complete U6 sequences. Integers represent transition; only when a suffix  is appended is a transversion indicated. Underlined nucleotide positions appear more than once in the tree. The rCRS is indicated.Click here for file


As a group of highly conserved small non-coding RNAs with a length of 21~23 nucleotides, microRNAs (miRNAs) regulate the gene expression post-transcriptionally by base pairing with the partial or full complementary sequences in target mRNAs, thus resulting in the repression of mRNA translation and the acceleration of mRNA degradation. Recent work has revealed that miRNAs are essential for the development and functioning of the skeletal muscles where they are. In particular, miR-206 has not only been identified as the only miRNA expressed in skeletal muscles, but also exhibited crucial roles in regulation of the muscle development. Although miRNAs are known to regulate various biological processes ranging from development to cancer, much less is known about their role in the dynamic regulation of the mammalian circadian clock.A detailed dynamic model of miR-206-mediated mammalian circadian clock system was developed presently by using Hill-type terms, Michaelis-Menten type and mass action kinetics. Based on a system-theoretic approach, the model accurately predicts both the periodicity and the entrainment of the circadian clock. It also explores the dynamics properties of the oscillations mediated by miR-206 by means of sensitivity analysis and alterations of parameters. Our results show that miR-206 is an important regulator of the circadian clock in skeletal muscle, and thus by study of miR-206 the main features of its mediation on the clock may be captured. Simulations of these processes display that the amplitude and frequency of the oscillation can be significantly altered through the miR-206-mediated control.MiR-206 has a profound effect on the dynamic mechanism of the mammalian circadian clock, both by control of the amplitude and control or alteration of the frequency to affect the level of the gene expression and to interfere with the temporal sequence of the gene production or delivery. This undoubtedly uncovers a new mechanism for regulation of the circadian clock at a post-transcriptional level and provides important insights into the normal development as well as the pathological conditions of skeletal muscles, such as the aging, chronic disease and cancer. Most organisms use circadian rhythms to keep the temporal order and anticipate daily variations in their environmental changes . CircadiAt the molecular level, molecular and genetic studies indicate that a circadian period arises from a system of interconnected feedback loops which control the transcription of a small number of "clock" genes ,7. It isMiRNAs are small non-coding RNAs, with approximately 22 nucleotides long that are involved in post-transcriptional regulation of the gene expressions in most living organisms. They are well conserved in eukaryotic organisms and are thought to be vital and evolutionarily ancient components of the genetic regulation . MiRNA mLately, a large fraction of miRNAs were found exhibiting strict developmental stage and tissue-dependent expression manner which is critical for their appropriate activities , and theIt is known that circadian clock system involves many components and complex interactions in living organisms . ClearlyHowever, up to now, little is known about the contribution effects of the translational controls to the circadian rhythms system. No further model for the mammalian circadian clock involved in the miRNA-mediated regulation mechanism has been developed, particularly for the miR-206, a critical fine-tuner of the core circadian clock. To address this issue and in particular to explore the translational control by miRNAs, in the present work we attempt to establish a detailed quantitative mathematical model for the circadian rhythms oscillators in mammalian clock, which involves a Clock-Myod1-miR-206 feedback loop controlling the timing of the circadian cycle. The process includes several aspects. Firstly, the generation of the basic circadian rhythm in our model depends on two negative autoregulations of the gene expression. One is the negative autoregulatory feedback exerted by the CLOCK-BMAL1 on the expression of Bmal1 gene. The other is the Clock expression which is subjected to a negative autoregulation by the CLOCK protein. Secondly, besides the negative autoregulatory feedback of the gene expression, the interplay of the negative and positive feedback loops is also taken into consideration. Actually, these interlocked feedback loops potentially allow for multiple inputs and outputs at different phases  and helpRecent studies showed that the molecular mechanism of circadian oscillations not only relies on the feedback loops of the gene expression, but also is affected by the interfering of miRNAs in the circadian rhythm choreography . TherefoUsing a set of parameters with appropriate biological values, the modeling work was carried out presently Table  by numerBM), Myod1 mRNA (myM) and Clock mRNA (PM) are all kept with a period of ~24 h , Bmal1 mRNA (BM), cytosolic and nuclear CLOCK proteins  and the complex between the nuclear CLOCK and BMAL1 proteins (NI), only small variations in the amplitude of the components are observed without the miR-206 regulation. But for other components, like the cytosolic CLOCK proteins (1P), Myod1 mRNA (myM) and cytosolic MYOD1 (CMY), their expression levels are largely enhanced in the absence of miRNA. This may be due to the fact that MyoD is a direct target of the circadian transcriptional activators NI, which binds in a rhythmic manner to the core enhancer of the MyoD promoter [N Idirectly controls the my Mand C MYexpression, which is more important than the indirect miRNA regulation. These results may be related to the fact that the transcription and translation of key circadian core components, on one hand are tightly connected with each other, and on the other hand may buffer each other. Such a regulatory feature could also explain why a major increase in the transcription, like the one caused by the myod1 gene, results in only a modest increase in Myod1 mRNA abundance and probably an even more modest increase in the translated protein.The same tendency observed in Figure promoter . TherefoPM, BM, 0P, 2P, N Pand NI). But the expression levels of cytosolic CLOCK proteins, Myod1 mRNA and cytosolic MYOD1 are largely enhanced in the absence of miRNA, reflecting the fact that the complex NI's direct effects on the my Mand C MYexpression is more important than the indirect miRNA regulation. These observations may provide a plausible mechanism through which tissue-specific factors such as Myod1 and miR-206 can convey unique tissue requirements to the circadian clock. 3) Once again, it is confirmed that miR-206 is necessary for accurate circadian timekeeping, since in the absence of miRNA, the oscillation period of the system as shown by the three representative components, Bmal1, Myod1 and Clock mRNAs and their corresponding proteins is not very constant and changes with time. At 48 h, the period is 24.5 h. But when the simulation time increases to 300 h, the period has changed up to ~24.6 h, and even up to ~24.7 h at 1000 h or much longer time. However the system is more stable in the presence of miRNA when the period of the oscillatory system is always 24.0 h, which further indicates the important role of miR-206 plays in the accurate circadian timekeeping.From the above results, three interesting findings are presented: 1) Although the miR-206 regulation is crucial in controlling the dynamics of the circadian rhythms system, it is not strong and fits exactly some recent data demonstrating that a Drosophila miRNA can function as a buffering agent against the environmental perturbations during the development . 2) SustHaving many successfully applications in the study of biochemical systems ,43, senssBV) has the largest impact on the whole system, affecting 21 out of the total 49 reactions while mB Vsignificantly affects 20 reactions, sB Kaffects 14 reactions, and 1B, V3B Vand I Krespectively affecting 13 reactions  reflects a fact that miRNA though regulating the system in a relatively weak manner, is still crucial in controlling the dynamics of the circadian clock.In addition, another interesting finding is that parameter nB, PM, N Pand MiR-206. These curves were obtained when C1 = 0.23 nM·h-1 and all other parameters remained as the same values as in Figure As we know, sustained oscillations only occur within an appropriate range of parameter values, and beyond this range the system tends to reach a steady state. Such an evolution is often associated with the occurrence of damped oscillations. The mammalian circadian system is thought to be composed of a hierarchical set of oscillators with the suprachiasmatic nuclei acting as a master pacemaker ,44 whichsBV) exhibits the largest effects on our system, some explorations on variation of the parameter space were carried out presently. The value of sB Vwas changed as 0.14, 0.28  and 0.56 nM·h-1, when values of other parameters were kept fixed. Figure sB V= 0.14, 0.28 and 0.56 nM·h-1, respectively. When sB Vdecreases from 0.28 to 0.14 nM·h-1, the oscillations of Bmal1 mRNA , indicating that the Clock is robust to the variations of sBV. This on one hand might explain why the biological clock period is always constant in various organisms, and on the other hand is also consistent with the fact that the Clock expression is necessary to maintain the normal circadian rhythmicity [In contrast, the oscillation of Clock mRNA keeps constant with only slight variations occurring in the amplitude and period with the change of B Figure . This isthmicity . Clearlythmicity .sB Vvalue when keeping all other rate constants fixed . Therefore, an attempt has been made to investigate how the changes of the kinetic parameters associated with miR-206 cause changes in the dynamic behavior of the biological clock system.Previous research suggested that miR-206 is specifically expressed in skeletal muscle , which iC1 was set to 0.0, 0.01 and 1.0 nM·h-1, with all other rate constants kept fixed. The obtained results are shown in Figure C1 = 0.01 and 1.0 nM·h-1 respectively. As seen from this figure, when C1 = 0.0 nM·h-1, the system still keeps relatively constant oscillations in the absence of miRNA, which might indicate that the regulation of miR-206 in the biological clock system is relatively weak [C1 = 0.01 nM·h-1), the oscillatory behavior of the system appears in an extremely constant manner. When C1 changes from 0.0 to 0.01 nM·h1, the amplitudes of oscillation for Bmal1 and Clock mRNAs do not show any significant changes, but the amplitude of oscillation for Myod1 mRNA decreases from 0.5 to 0.3 nM. The reason is that Myod1 directly activates the expression level of miR-206, thus resulting in the difference of expressions between Bmal1 and Myod1. All these results reveal that the sustained oscillations occur only within a certain range of parameter values. Beyond this range, the system tends to a steady state. So when more miRNA is produced (C1 = 1 nM·h-1) in the system, all the above three mRNAs evolve to a steady state and no oscillations occur. Clearly the over expression of miR-206 in the system disturbs the biological clock and makes the periodic oscillations collapsed. This result indicates that miRNAs are important in maintaining the tissue and cell normal function, and the abnormal expression of miRNAs may lead to unexpected diseases.It is also interesting to explore the variation of the frequency and amplitude when ely weak . But oncC1, indicating that the temporal sequence of the gene production can be interfered , C3 (the formation rate of the RISC complex), C4 (the maximum degradation rate of the ISC Rcomplex) and sC K(the synthesis rate of MYOD1) on the whole system are also investigated, respectively. Figure C2 and C3 are increased by 10 folds compared to their normal values (Table C4 and sCK) also do not significantly affect the system, and the observed evolutionary curves with different parameter values are almost totally overlapped with each other. These results demonstrate that all the four parameters are not as significant as C1, the miRNA production rate, to the oscillator system, which is consistent with the findings obtained from the sensitivity analysis.In addition, the effects of the variation of parameters MiRNAs play an important role in various biological functions and represent a potentially novel avenue by which biological timing processes can be regulated. In this paper, the dynamics of regulation by miR-206 were investigated based on the Clock-Myod1-miR-206 interacting positive and negative feedback loops controlling the timing of the circadian cycle in mammalian skeletal muscle. To this end, a system-theoretic approach by using Hill-type terms, Michaelis-Menten type and mass action kinetics is introduced in this work and the dynamics is implemented deterministically. The model outlined presently not only introduces miR-206 into the field of circadian timing, but also accurately predicts the clock periodicity and clock entrainment. A sustained periodic circadian oscillation occurs in the circadian clock system when the series of parameters were set with biologically reasonable values. In addition, this model reveals the possible existence of multiple sources of biological oscillatory behavior, and provides a plausible mechanism through which tissue-specific factors such as Myod1 and miR-206 can convey unique tissue requirements to the circadian clock. In addition, the results point out the important effects of parameter variations such as the miR-206 synthesis on the regulation of the mammalian clock system, which not only changes the amplitude but also alters the frequency of the oscillations of the deterministic system. In other words, miR-206 plays a significant role on the dynamics of regulatory systems, both by affecting the level of the gene expression and by interfering with the temporal sequence of the gene production or transmission.In short, the essential qualitative features of these results indicate that the miRNA regulation may be one of the primary means for controlling the period of oscillatory molecules and biochemical pathways within mammalian circadian clock. We provide testable hypothesis for experimental biologists to further investigate miRNA's functional roles in regulating cellular processes and development. The challenge for future studies will be a focus on the modulation of the miRNA expression as a potentially powerful diagnostic and therapeutic approach to treat cardiac and skeletal muscle disease.In this work, a model of the mammalian circadian core oscillator which is mediated by miRNA was developed, using a series of ordinary differential equations (ODEs) Figure . The focEvidence to support the model is as follows:my Mand BM). Then these mRNAs are transported to the cytoplasm and degraded.1) In the cell nucleus, the genes of Myod1 and Bmal1 are transcribed into the corresponding Myod1 and Bmal1 mRNAs (denoted C MYand cB).2) In the cytoplasm, the two mRNAs are translated into unphosphorylated proteins, i.e., the MYOD1 and BMAL1 (denoted by cp MYand cpB) and degradation processes.3) These unphosphorylated proteins further undergo reversible phosphorylation .4) The cytosolic BMAL1 protein is reversibly transported into the nucleus. In turn, the nuclear BMAL1 protein proceeds reversible phosphorylation and then degrades in nucleus (with unphosphorylated and phosphorylated BMAL1 denoted by PM), which transports into the cytoplasm where it is then translated into the CLOCK protein (P0) and degrades. In the cytoplasm, the CLOCK protein can be reversibly phosphorylated from the form of P0 into P1 and P2, successively.5) The model also incorporates the Clock gene transcription, i.e., the Clock mRNA (P2 is degraded and transported into the nucleus (NP), and the nuclear form of protein N Psubsequently represses its gene expression and produces a negative feedback control loop .6) The latter form p Figure , Loop 2 7) In the nucleus, the BMAL1-CLOCK heterodimer increases the rate of transcription of the corresponding gene, Myod1 . At the ISCR) and then degrades [8) In turn, the Myod1 gene, which is known to regulate the miR-206 expression  has beendegrades .In summary, this model displays the essential components and characteristics of the circadian clock, and provides a plausible mechanism through which the tissue-specific factors such as Myod1 and miR-206 can convey unique tissue requirements to the circadian clock. The detailed pathway is shown in Figure Deterministic models based on ODEs can provide insights into the understanding of the biological pathway, as how the overall behaviors depend on a set of constituent reactions. The whole circadian clock pathway is modeled using a set of ODEs which were numerically solved by stiff method. The variables of the system represent the concentrations of each species like mRNAs or proteins Figure .In this paper, we develop a detailed distinctly dynamic model of the mammalian circadian clock mediated by miR-206 by using Hill-type terms, Michaelis-Menten type and mass action kinetics, which are the mathematical models that explain and predict the behaviors of the molecules in dynamic equilibrium. The time evolution of the model is governed by a system of 19 kinetic Equations (1)-(19). The ODEs of the dynamic model are expressed as follows:(a) genes of Bmal1, Clock and Myod1:Although Eqs. (1) and (2) are applied for different genes, the underlying principles and formulations are similar: the first two terms represent the generation and degradation of given genes, while the last one represents the inhibition of gene transcription by the negative feedback control ,55.N I[In Eq. (3), the first two terms are the same to Eqs. (1) and (2). The last term represents the activation of the gene transcription by the complex IN .(b) mRNAs of Bmal1, Myod1 and Clock:B Mand my Mas the concentrations of mRNA product at time t, and then the variations of the two mRNAs with time are summarized by Eqs. (4) and (5). The two equations describe the synthesis, maximum degradation and nonspecific degradation of the Bmal1 mRNA and Myod1 mRNA, respectively.We define P M(the mRNA of Clock). The third term represents the formation of the miR-206-P M(ISCR) complex.The first two terms in Eq. (6) are similar to Eq. (5) by representing the production and degradation of (c) reversibly phosphorylated and non-phosphorylated proteins MYOD1 in the cytoplasm:where the phosphorylation and dephosphorylation in the first two terms of each equation are governed by the Michaelis-Menten kinetics. The third term in above two equations represents the nonspecific degradation of the non-phosphorylated and phosphorylated cytosolic MYOD1s, respectively. The last term in Eq. (7) represents the synthesis of the cytosolic MYOD1, while in Eq. (8) represents the maximum degradation of the phosphorylated cytosolic MYOD1 using the Michaelis-Menten kinetics, respectively.(d) phosphorylated and non-phosphorylated proteins BMAL1 and CLOCK in the cytoplasm and nucleus:These two equations describe the synthesis and degradation of the cytosolic and nuclear BMAL1 proteins, respectively. The first two terms are similar to Eqs. (7) and (8), while the remaining terms correspond to the synthesis or degradation of the proteins by using the mass action rules.Eqs. (11) and (12) describe the synthesis and degradation of the phosphorylated cytosolic or nucleus BMAL1 proteins, respectively. The principles for the two equations are the same as Eq. (8).Equations (13)-(15) relate to the synthesis and degradation of phosphorylated and non-phosphorylated cytosolic CLOCK proteins. Eq. (16) refers to the variation of the concentration of nucleus CLOCK protein with time. The principles are similar to the above equations.(e) BMAL1- CLOCK complex in nucleus:NI, while the last three terms represent the dissociation, maximum degradation and nonspecific degradation of NI, respectively.where the first term corresponds to the synthesis of complex ISCR:(f) miR-206 and ISC Ris given by Eq. (19). These two equations are similar in that they describe the synthesis and degradation of miR-206 and ISC Rrespectively, but different in that the synthesis of miR-206 is described by the Hill-type function.The concentration change of miR-206 is given by Eq. (18) and that for c, n, cp or np for the cytosolic, nuclear, cytosolic phosphorylated or nuclear phosphorylated terms, respectively.The definition of all the parameters in Eqs. 1)-(19) is shown in Table -(19) is AS) is that A Sstudies the relationships between the information flowing in and out of a model. It is a step in the modeling process aiming at identifying the important uncertainties for the purpose of prioritizing the additional data collection or research. It is used to determine how "sensitive" a model is to those changes occurring in the parameter values or in the structure of the model. A dynamic sensitivity value reflects a relative relationship between the magnitudes of a parameter (input) and a state variable (output) at any time. Thus, the measure of the sensitivity is that the sensitivity of an item is taken to be the output with respect to a single parameter iX:The definition of sensitivity analysis  relative to the parameter iX, and the sensitivity of each variable corresponds to a specified parameter. In order to obtain the scaled sensitivity coefficient A Sthat is dimensionless and plotted for maintaining the visual presentation drawing, the results are normalized.The quantity The authors declare that they have no competing interests.Conceived and designed the experiments: YW, YL. Performed the experiments: WZ. Analyzed the data: WZ, XW, LW. Wrote the paper: WZ, YL. All authors have read and approved the final manuscript.