Calcifying organisms have exhibited reduced calcification under lower saturation state conditions in aquaria. However, the in situ sensitivity of calcifying ecosystems to future ocean acidification remains unknown. Here we assess the community level sensitivity of calcification to local CO2-induced acidification caused by natural respiration in an unperturbed, biodiverse, temperate intertidal ecosystem. We find that on hourly timescales nighttime community calcification is strongly influenced by Ωarag, with greater net calcium carbonate dissolution under more acidic conditions. Daytime calcification however, is not detectably affected by Ωarag. If the short-term sensitivity of community calcification to Ωarag is representative of the long-term sensitivity to ocean acidification, nighttime dissolution in these intertidal ecosystems could more than double by 2050, with significant ecological and economic consequences.Anthropogenic emissions of carbon dioxide (CO Our intertidal study site at Bodega Marine Reserve in Northern California experiences extreme variation in calcium carbonate saturation state at low tide due to photosynthetic activity and respiration occurring after the time at which the pools become isolated from the open ocean. As photosynthetic activity is largely dependent on temperature and photosynthetically active radiation (PAR), which vary on a diurnal timescale, whereas tide pool isolation is predominantly a function of tidal phase, we were able to separate the influence of calcium carbonate saturation state on calcification from the influence of temperature and PAR. This system therefore provides a unique opportunity to characterise the Corallina vancouveriensis and Calliarthron sp.; 10.1–37.3% benthic cover) and crustose coralline algae . The tide pool communities included various non-calcifying red algae , brown algae , green algae  and seagrasses . We note that pools also contained a diverse calcifying invertebrate community, including bivalves  and gastropods . In the majority of pools, the dominant component of calcifying invertebrate biomass was Chlorostoma funebralis  and dissolved inorganic carbon (CT) were highest before sunrise  and decreased through the course of the day to minimum values in the late afternoon . The pCO2 showed similar declines, with a peak pre-sunrise value of 3276μatm and a minimum value of <10 μatm by mid-to-late afternoon. The pH, CO32− and Ωarag all show large concurrent decreases through the night and increases during the day, with pH increasing from a minimum value before sunrise of 7.22 to a maximum late afternoon value of 9.00, and Ωarag increasing through the day from a minimum value of 0.38 before sunrise to a maximum of 8.43. As discussed in greater detail below, the variation in carbonate chemistry parameters can be largely attributed to patterns of calcification, net primary production and changes in tide pool temperature.The carbonate chemistry of seawater in the tide pools varied substantially throughout the day in all pools studied . Total aAT and CT  shows similar diel trends to net community production (Pnet), with peak rates typically occurring between 11:00 and 13:00 . Gnet is linearly correlated to PAR, achieving statistical significance (p < 0.01) in all pools except for Pool 4 (p = 0.11). This is likely an artefact of the fewer measurements made at low light levels in Pool 4. While linearly correlated, the data suggest that the relationship between Gnet and PAR can be better described by a Michaelis-Menten function with the positive influence of PAR on Gnet saturating at PAR values >1000 μmol m−2 s−1 (net is linearly correlated to temperature (p < 0.05) and Ωarag (p < 0.05) in all pools with the exception of Pool 4.In all pools, G m−2 s−1 . Gnet isnet, PAR, and a Gaussian function of temperature (Tf) explain 33–71% of the variation in daytime Gnet in the tide pools studied  is found to be strongly influenced by Ωarag, with higher nighttime Gnet at higher Ωarag values in all tide pools. This is in contrast to the Ωarag insensitivity described for daytime Gnet. Nighttime Gnet typically occurs at lower Ωarag values (0.38–2.06) than those present during the day (0.56–8.43), and is predominately negative, indicating net CaCO3 dissolution . Daytime calcification rates could exhibit some dependency on Ωarag as has been shown for individual species in laboratory manipulations that encompass a similar Ωarag range1012arag and other potential explanatory variables such as PAR, this dependence may not be detectable in situ18The finding that Ω account , suggestnet and Pnet during the daytime imply that calcification and photosynthesis are intimately linked in these tide pools. This suggests that photosynthesis could enhance calcification processes during daylight hours20arag. In the intertidal communities studied here, photosynthetic calcifiers  may have a larger influence on carbonate chemistry dynamics than non-photosynthetic calcifying species, such as molluscs, during daylight hours. While we did not assess the extent to which individual taxa contribute to net community calcification in the tide pools, our results allow for predictions about the vulnerability of net daytime community calcification to variability in Ωarag across a range of community compositions.The positive correlations between Garag as projected by an ensemble of current-generation Earth System Models, is highly dependent on the economic and environmental pathway taken. Under a business-as-usual scenario (RCP 8.5), global mean Ωarag is projected to decline by 1.32 by the year 2100, relative to global mean Ωarag in 1990–2000. Even under an extremely ambitious pathway aimed to limit global mean temperature rise to 2 °C (RCP 2.6), which assumes full participation in emission reductions by all countries, and the possibility of negative emissionsarag is still expected to decline by up to 0.33 throughout the 21st Century, relative to 1990–2000 mean Ωarag. Although the extent to which global declines in Ωarag are reflected in intertidal zones will vary regionally and be mediated by local processesarag are considerably smaller than our measured diel variability due to photosynthesis and respiration. The impact of ocean acidification in intertidal ecosystems will therefore be a consequence of the sensitivity of communities already exposed to a wide Ωarag range experiencing a relatively smaller decline in mean background ΩaragAs shown in arag, with Pnet and a Gaussian function of temperature (Tf) found to offer additional explanatory power in certain tide pools. Ωarag coefficients in these models are between 4.17 ± 1.11 and 8.70 ± 1.66 depending on the tide pool. Therefore, a decline in background Ωarag values of 0.5, which is highly likely by 2050 under even optimistic scenarios of future climate change  analysis.Our study site consisted of 4 rocky tide pools located in Horseshoe Cove, Bodega Marine Reserve, California , pH, dissolved inorganic carbon (CT and nutrients (NO2− + NO3− and NH3 + NH4+). AT was measured using a Metrohm 855 autotitrator. Three replicates of each sample were run, and the mean value of the two closest replicates was used (<2 μmol kg−1 (1SD) instrument precision). Titrant was standardised using reference material supplied by the Dickson Lab (Batch 141). The pH and O2 values measured via CTD were calibrated using spectrophotometric pH measurements and Winkler titrations, respectively, conducted according to best practicesCT was measured by infrared adsorption via a LICOR7000 CO2/H2O analyser coupled with a custom-made sample delivery system built by Stanford University’s Stable Isotope Laboratory (<2 μmol kg−1 (1SD) instrument precision). Nutrients were measured photometrically using a WestCo SmartChem 200 discrete analyser (~0.05 μmol L−1 instrument precision). The pCO2, CO32− and aragonite saturation state (Ωarag) values were calculated with AT and CT for 2014 data, and AT and pH for 2015 data, using the CO2SYS program1 and K2 constants of Mehrbach et al.In total, 566 seawater samples were analysed for total alkalinity (http://bml.ucdavis.edu/boon/index.html). This record is measured within 100 m of the tide pools and is representative of local conditions. However, we note that local topography, water depth, and turbidity are additional influences on the direct PAR that tide pool communities received during the sample collection periods.Photosynthetically-active radiation (PAR) data were provided by the Bodega Ocean Observing Node, University of California, Bodega Marine Laboratory  was calculated in each tide pool using the salinity-normalised alkalinity anomaly methodNet community calcification , d is the mean tide pool depth (m), ρ is the density of sea water (kg m−3)Δt is the time period (h). Net community production  is calculated as:where ΔCT is the change in salinity normalised CT (μmol kg−1) and −1 m−2 h−1) is the air-sea flux of CO2 calculated using the wind speed-dependent gas transfer function of Ho et al. 20062 according to Weiss 19742 concentration of 401 ppm and 403 ppm for the 2014 and 2015 sampling periods respectively (the NOAA/ESRL Mauna Loa mean CO2 concentration over the sampling periods). Wind speed data were provided by the Bodega Ocean Observing Node, University of California, Bodega Marine Laboratory.where Δnet, PAR, temperature, and Ωarag on the rate of community calcification (Gnet). Such an approach has been previously adopted in tropical environments arag anomalies are given for the Representative Concentration Pathways 2.6 and 8.539. RCP 2.6 and RCP 8.5 respectively represent the most extreme mitigation and business-as-usual RCP scenarios conducted in CMIP5 and are therefore considered to encompass the range of future potential Ωarag values.Coupled Model Intercomparison Project Phase 5 (CMIP5) global mean ΩHow to cite this article: Kwiatkowski, L. et al. Nighttime dissolution in a temperate coastal ocean ecosystem increases under acidification. Sci. Rep.6, 22984; doi: 10.1038/srep22984 (2016).


A 77-year-old female patient presented to the hospital’s emergency department with palpitations. The electrocardiogram shows a supraventricular bigeminy where the premature beats are due to aberrant conduction (wider and deeper S waves from lead V1 to V5 and ‘RSR’ morphology in lead V6) but the rhythm is not sinus (Fig. AnswerYou will find the answer elsewhere in this issue.


However, uncertainties remain regarding the use of δ2H, since little is known about the potential variation in the amount of exchangeable hydrogen (Hex) among common sample materials or the patterns of δ2H when entire food webs are considered. We assessed differences in Hex among the typical sample materials in freshwater studies and used δ2H, δ13C and δ15N to compare their effectiveness in tracing allochthonous matter in food webs of two small temperate lakes. Our results showed higher average amounts of Hex in animal tissues  compared to most plant material , with the exception of aquatic vascular plants . The amount of Hex correlated strongly with sample lipid content (inferred from C:N ratios) in fish and zooplankton samples. Overall, the three isotopes provided good separation of sources , particularly the δ2H followed by δ13C. Aquatic macrophytes revealed unexpectedly high δ2H values, having more elevated δ2H values than terrestrial organic matter with direct implications for estimating consumer allochthony. Organic matter from macrophytes significantly contributed to the food webs in both lakes highlighting the need to include macrophytes as a potential source when using stable isotopes to estimate trophic structures and contributions from allochthonous sources.Hydrogen stable isotopes (δ The trophic position of a consumer (t) can then be calculated from: t = 2 + ([δ15Ncons—δ15Npel—(δ13Clit—δ13Cpel) × BS × LFcons] / ΔN), where ΔN is the discrimination factor for δ15N. Fish were assumed to acquire allo-om indirectly through their prey and therefore mixing models of fish used invertebrates (for non-piscivorous fish) and invertebrates and prey fish  as prey sources. The estimates of allo-om contribution were then calculated using ratio calculation from each individual prey source  and terrestrial plants with an additional transpiration process are expected to be considerably elevated in their final δ2H values compared to aquatic plants. At present it is difficult to explain the observed δ2H patterns in aquatic vegetation  [2H values in both our lakes were considerably elevated compared to other sources of organic matter, even rather small amounts of organic matter derived from macrophytes can significantly bias the estimates, if only algae and terrestrial matter were assumed as sources. Therefore, macrophytes should be included as one source in mixing models for lakes where they are present .The importance of including macrophytes as one potential source of organic matter in the mixing models when estimating allochthonous contributions was highlighted in the comparison of models with and without macrophytes  , 54, indlzensee) . Our cal2H values. Since lipids have very low δ2H values, even small amounts of storage lipids can have significant impact on the bulk sample δ2H values. We did not remove lipids from our samples but in particular zooplankton is known to accumulate appreciable amounts of lipids, considerably affecting the isotope values analyzed from bulk samples [2H values of zooplankton and would have increased the allochthony in HCN mixing models. In addition, very little is known about the potential discrimination of H isotopes in trophic transfers from prey to consumers. In our mixing models we used the common practice of assuming no real trophic discrimination, but did assume ~17% contribution from environmental water at each trophic level [Estimates of consumer allochthony using either CN or HCN mixing models . In general, however, the results fit to expected ranges relatively well and align with those of most common standard materials (such as keratin). Nonetheless, we concur with previous studies [2H results before more laboratory and field studies on the effects of environmental water, lipids and δ2H patterns in ecosystems are available. Furthermore, the importance of aquatic macrophytes as a dietary source for lake consumers was detected more clearly when using δ2H. Due to the high δ2H values of macrophytes, estimates of allochthony by mixing models were largely dependent on whether macrophytes were included or not. We therefore strongly recommend future studies to include macrophytes as a potential resource when using δ2H with δ13C and δ15N in aquatic food web studies.Our results highlight the benefits of combining δ studies , 32–33 uS1 Dataset(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The break-up of Pangæa was principally facilitated by tensional plate stress acting on pre-existing suture zones. The rifting of Pangæa began during the Early Permian along the southern Tethys margin and produced the lenticular-shaped continent known as Cimmeria. A mantle-plume model is ascribed to explain the rift-related volcanism but the NW-SE oriented Cimmerian rifts do not correlate well with pre-existing suture zones or ‘structural heterogeneities’ but appear to have a pertinent spatial and temporal association with Late Palæozoic glacial-interglacial cycles. Mantle potential temperature estimates of Cimmerian rift-related basalts (1410 °C ± 50 °C) are similar to ambient mantle conditions rather than an active mantle-plume rift as previously suggested. Moreover, we find that the distribution of glacial deposits shows significant temporal and spatial concurrence between the glacial retreat margins and rifting sites. We conclude that the location and timing of Cimmerian rifting resulted from the exploitation of structural heterogeneities within the crust that formed due to repeated glacial-interglacial cycles during the Late Palæozoic. Such effects of continental deglaciation helped to create the lenticular shape of Cimmeria and Neotethys Ocean suggesting that, in some instances, climate change may directly influence the location of rifting. Crustal thinning, narrow rifting characterized by localization of strain through large boundary fault activation, and basin subsidence are the signature features of the early stages of continental break-up1232569The amalgamation and break-up of Pangæa exemplifies the secular nature of global tectonic processes as plate stresses transitioned from compressional to extensional during the Late Carboniferous to Early Jurassic61011115The break-up of Gondwana began during the Early Permian along a ~13,000 km long NW-SE oriented continental rift just as the Late Palæozoic continental ice sheet retreated16171920222319202122627A lenticular ribbon shape continent is not unique to Cimmeria as rifted peri-Gondwana terranes  had ribbon-shapes during the opening of Rheic Ocean26TP) required to generate the primary melt composition of basalt from within-plate or extensional settings can be calculated from their bulk composition. Thus the mafic rocks from Oman, Kashmir and Tibet can be used to deduce the regional thermal conditions during Early Permian rifting32To better understand the tectonomagmatic development of Cimmeria, we investigate the thermal regime of the basaltic rocks associated with rifting and the structural controls that led to the formation of its lenticular shape. Anomalously hot (>1550 °C) or ambient (1350 ± 50 °C) mantle potential temperatures  and relatively oxidizing conditions (Fe2O3/TiO2 = 1.0). The relatively reducing primary melt models produced compositions that range from high-Mg basalt to picrite (MgO = 12.1 wt% to 15.8 wt%) with corresponding eruption (T) and mantle potential (TP) temperatures between 1260 °C and 1360 °C and 1360 °C and 1460 °C respectively . Irrespective of the relative oxidation state used in the models, the results show that the temperature of the mantle that produced the primary magmas of the basalts was not anomalously hot but rather similar to ambient mantle conditions (1300 °C to 1400 °C).Basalts that fractionated plagioclase, pyroxene or both are not suitable for primary melt and ectively . The relNd(t) values of Cimmerian rift-related basalts. Some of the isotopic variability in the basalts is undoubtedly related to crustal contamination but there is no systematic spatial variation. The Oman, Qiangtang and Mojiang basaltic rocks tend to have more depleted εNd(t) signatures than the Panjal and Tibet basalts implying local mantle sources (sub-lithospheric vs. lithospheric) were likely responsible for their genesis. Moreover, the TP values are not sufficiently different to distinguish a regional thermal trend that would be indicative of a mantle-plume regime 414243Mantle-plumes are thought to have a diameter of ~2500 km as they flatten-out near the top of the mantleThe northward directed slab pull under the E-W trending subduction zones along the northern margin of the Palæotethys is considered to be the main driving mechanism that generated the NW-SE trending ‘Cimmerian rifts’ 1112, yet11123031The Late Palæozoic is characterized as a time of large continental and alpine glaciers that covered the Polar regions of southern Gondwana471 − ơ3) to be 18.6 MPa km−1  5.58 MPa km−1 , Southern Qiangtang (Tibet), and Australia is evidence that this region experienced Late Palæozoic glaciation during the latest Carboniferous to earliest Permian17TP > 1550 °C). Although no inherent structural heterogeneity zones or icosahedral tessellation boundaries were present for rift margins to act upon, it is likely that a large differential stress environment, strong postglacial rebound stress, and the shielding effect of the ice sheet created regions of structural heterogeneities for a later extensional tectonic stress to exploit. Therefore, it is very likely that the eruption of flood basalts, rifting of Cimmeria and the formation of the Neotethys Ocean are directly related to deglaciation of the Late Palæozoic ice sheet. Such a relationship between environmental change and supercontinent break-up may be unique to the Late Palæozoic.There is a spatial and temporal association between glacial retreat and rifting of Cimmerian terranes from Gondwana during the Late Palæozoic. Ambient mantle potential temperatures estimates of Early Permian Cimmerian rift-related basalts from Oman, Kashmir and Tibet are supportive of an archetypical passive continental rift rather than an active mantle-plume rift with anomalously hot mantle  results are reported in Dataset S2.The primary melt compositions and mantle potential temperature estimates were calculated using PRIMELT3How to cite this article: Yeh, M.-W. and Shellnutt, J. G. The initial break-up of Pangaea elicited by Late Palæozoic deglaciation. Sci. Rep. 6, 31442; doi: 10.1038/srep31442 (2016).


Bhirrana was part of a high concentration of settlements along the dried up mythical Vedic river valley ‘Saraswati’, an extension of Ghaggar river in the Thar desert. Isotope and archaeological data suggest that the pre-Harappans started inhabiting this area along the mighty Ghaggar-Hakra rivers fed by intensified monsoon from 9 to 7 ka BP. The monsoon monotonically declined after 7 ka yet the settlements continued to survive from early to mature Harappan time. Our study suggests that other cause like change in subsistence strategy by shifting crop patterns rather than climate change was responsible for Harappan collapse.The antiquity and decline of the Bronze Age Harappan civilization in the Indus-Ghaggar-Hakra river valleys is an enigma in archaeology. Weakening of the monsoon after ~5 ka BP (and droughts throughout the Asia) is a strong contender for the Harappan collapse, although controversy exists about the synchroneity of climate change and collapse of civilization. One reason for this controversy is lack of a continuous record of cultural levels and palaeomonsoon change in close proximity. We report a high resolution oxygen isotope (δ The rise of the post-Neolithic Bronze Age Harappan civilization 5.7–3.3 ka BP  spread along the Indus Valley of Pakistan through the plains of NW India, including into the state of Gujarat and up to the Arabian Sea and its decline has remained an enigma in archaeological investigation123457810610111418O) record of animal teeth and bone phosphates (bioapatites) from an excavated archaeological trench at Bhirrana, state of Haryana, NW India, to reconstruct a paleomonsoonal history of the settlement site itself. Based on radiocarbon ages from different trenches and levels the settlement at Bhirrana has been inferred to be the oldest (>9 ka BP) in the Indian sub-continent17Potential reasons for these conflicting interpretations is that the climate reconstructions were made from locations  distant from the main Harappan settlement areas or that the climate proxies  could have been influenced by multiple local parameters apart from mere rainfall or temperature. To date no continuous climate record has existed close to or from the Harappan settlements. Here we report a high resolution bulk oxygen isotope δ1O record 14C dates from the site of Harappa and nearby localities. These periodization is temporally correlatable with the Indus valley civilisations from Baluchistan and Helmand province proposed by Shaffer22232526Archaeological chronologies of Harappan (Indus) civilization in South Asia2161319Based on the spatio-temporal distribution of the archaeological remains spread throughout the subcontinent a much older chronology has, however, been advocated by Possehl28303132893414C age of 8.35 ± 0.14 ka BP , Early Harappan (~8–6.5 ka BP), Early mature Harappan (~6.5–5 ka BP) and mature Harappan (~5–2.8 ka BP817182081717817173217Bos indicus), buffalo , goat (Capra hircus) and sheep (Ovis aries) from the earliest levels. Besides the dietary use of cattle and goats, wild fauna such as nilgai (Boselaphas tragocamelus), Indian spotted deer (Axis axis) and antelope (Antilope cervicapra) were also a part of the diet373839A compilation of calibrated radiocarbon dates of the charcoal samples and OSL dates of pottery (see later discussion) from various cultural levels of Bhirrana , retrieved during the excavation of 2005, is given in SI814C ages) proposed by earlier workers81718For retrieving information on past climatic changes we isotopically analysed bulk (see SI text) teeth and bone phosphates, wherever available, from the trench YF-2 which has both stratigraphic and sampling continuity . To chec18O  composition of fossil bone or tooth enamel bioapatite [carbonated hydroxyapatite414243444518O analysis of bioapatites are given in SI text. Under a constant body temperature of ~37 °C, the δ18O in mammalian phosphate (δ18Op) essentially depends on the δ18O value of water (δ18Ow) ingested by the organism. Between the water and phosphate, oxygen isotope is fractionated in two steps, i.e., between environmental and body water and between body water and phosphate in teeth and bones4718Op and δ18Ow414950ibid). For the Bhirrana mammals we used the taxon specific herbivorous mammal equations of Bryant and Froelich18Op data of bioapatites and calculated δ18OW are given in δ18OW variation as a function of depth and against Harappan chronology at Bhirrana proposed by Rao et al.18O in large mammals reflects the integrated mean annual δ18O of local meteoric water ingested by the animal during its life time. At several cultural levels we analysed multiple samples of either teeth or both teeth and bones. The spread in estimated δ18OW ranges from <1‰ to maximum ~4‰ and are probably due to the seasonal variation in δ18OW5253545518OW value from successive layers, we sampled bulk enamel or phosphate along the entire length of a single tooth or a bone (see SI text), yet the inter-sample seasonal signature might have been preserved in some cases. In spite of the inter-sample spread, the mean δ18OW values . The δ18OW values rapidly decreases towards the early Harappan phase reaching δ18O minimum of −9.01‰ at ~8 ka (trench depth ~308 cm). Thereafter the δ18OW monotonically gets enriched from early Harappan through early mature Harappan to mature Harappan, a time span from ~8 ka BP to 2.8 ka BP. We interpret this δ18OW variation through all the cultural levels at Bhirrana as major change in monsoonal precipitation during the last 9.5 ka. We compare the Bhirrana record with available monsoon records from Arabian Sea , lower oceanic salinity, increased upwelling, reduction in δ18O in speleothems from Arabia to Tibet, higher erosion rate in the Himalayas, and increased sedimentation in the Ganges deltaic plains  to −15‰ in peak monsoon periods (depletion) with weighted mean annual δ18O value of −7‰. The large monsoon depletion in δ18O results from well-known amount effect where excess rainfall is known to produce extreme depletion -Hakra (in Pakistan) river, referred to as mythical Vedic river ‘Saraswati’  originat7931332969th millennium BP, it continued and flourished in the face of overall declining rainfall throughout the middle to late Holocene period112373747576777816828384Study of fluvial morphodynamics coupled with detrital zircon analysis of river channel sands indicated presence of a more energetic fluvial regime before 5 ka across the entire Harappan landscape, stabilized alluvial systems during early Harappan 5.2–4.6 ka BP) and drying up of many river channels during post-Harappan period5 ka BP an7172How to cite this article: Sarkar, A. et al. Oxygen isotope in archaeological bioapatites from India: Implications to climate change and decline of Bronze Age Harappan civilization. Sci. Rep.6, 26555; doi: 10.1038/srep26555 (2016).


While this parameter did not exceed 2.1×104 cells mL−1 in the effluent from any water treatment plant, 50% of all the network samples contained more than 1.06×105 cells mL−1. This indisputably demonstrates biological instability in this particular drinking water distribution system, which was ascribed to a loss of disinfectant residuals and concomitant bacterial growth. The study highlights the potential of using cultivation-independent methods for the assessment of chlorinated water samples. In addition, it underlines the complexity of full-scale drinking water distribution systems, and the resulting challenges to establish the causes of biological instability.The purpose of a drinking water distribution system is to deliver drinking water to the consumer, preferably with the same quality as when it left the treatment plant. In this context, the maintenance of good microbiological quality is often referred to as biological stability, and the addition of sufficient chlorine residuals is regarded as one way to achieve this. The full-scale drinking water distribution system of Riga (Latvia) was investigated with respect to biological stability in chlorinated drinking water. Flow cytometric (FCM) intact cell concentrations, intracellular adenosine tri-phosphate (ATP), heterotrophic plate counts and residual chlorine measurements were performed to evaluate the drinking water quality and stability at 49 sampling points throughout the distribution network. Cell viability methods were compared and the importance of extracellular ATP measurements was examined as well. FCM intact cell concentrations varied from 5×10 The goal of public drinking water supply systems is to produce water of acceptable aesthetic and hygienic quality and to maintain that quality throughout distribution until the point of consumption. From a microbiological perspective, the quality of treated water can deteriorate as a result of excessive bacterial growth, which can lead to problems such as a sensory deterioration of water quality  as well as pathogen proliferation The concept of biological stability and its impact on a system's microbiology has been discussed extensively in the framework of non-chlorinated drinking water distribution systems In the present study we examined some of the above-discussed concepts in a full-scale, chlorinated distribution system in the city of Riga (Latvia) with a number of microbiological methods. The purpose was a detailed investigation of the entire city's distribution network, asking the basic question whether evidence of spatial and/or temporal biological instability exists, and if so, to which degree. Additional goals were to evaluate the use of fluorescent staining coupled with FCM, as well as ATP analysis, for the assessment of chlorinated drinking water in a distribution network with disinfectant residuals.Permission for sampling at all locations in the present study was obtained from the local water utility (Rīgas 3 d−1). Only the three major WTP, which are continuously operated, were included in the sampling campaign. Average WTP effluent water quality parameters for each treatment plant are shown in Sampling was performed in the full-scale distribution network of Riga (Latvia) with a total length of about 1400 km. The city is supplied with drinking water from six water treatment plants (WTP) produced from both surface and groundwater (150 000 m−1) for no more than 60 s, then immediately adjusted to a low velocity of 0.015–0.25 m s−1 and connected to an online system for monitoring pH, temperature, redox potential, electro-conductivity and turbidity. The low sampling velocity was specifically used to ensure a minimal possible impact of cell wall erosion and detachment from biofilms on the samples and measurements. Readings of all parameters were taken at 5–10 minute intervals, and water was only sampled for microbiological analysis once all of the parameters stabilized. The impact of this hydrant flushing is demonstrated in an example in A specific sampling protocol was designed and followed in order to avoid artifacts due to water stagnation in unused fire hydrants. Each hydrant was pre-flushed at a high velocity  and subsequent formation of a red compound at pH 6.2–6.5. Afterwards titration by means of a standard solution of ammonium iron (III) sulfate until disappearance of the red color was performed. Determination of total chlorine was performed according to EN ISO 7393-1, based on the reaction with DPD in the presence of an excess of potassium iodide, and then titration as described above.® Green I (SG) stock  was diluted 100x in anhydrous dimethylsulfoxide (DMSO) and propidium iodide  was mixed with the SYBR® Green I working solution to a final PI concentration of 0.6 mM. This working solution was stored at −20°C until use. From every water sample, 1 mL was stained with SGPI at 10 µL mL−1. Before analysis, samples were incubated in the dark for 15 minutes. Prior to FCM analysis, the water samples were diluted with 0.22 µm filtered bottled water  to 10% v/v of the initial concentration. FCM was performed using a Partec CyFlow SL instrument , equipped with a blue 25 mW solid state laser emitting light at a fixed wavelength of 488 nm. Green fluorescence was collected at 520±10 nm, red fluorescence above 630 nm, and high angle sideward scatter (SSC) at 488 nm. The trigger was set on the green fluorescence channel and data were acquired on two-parameter density plots while no compensation was used for any of the measurements. The CyFlow SL instrument is equipped with volumetric counting hardware and has an experimentally determined quantification limit of 1000 cells mL−1Staining and FCM analysis was done as described previously Total ATP was determined using the BacTiter-Glo reagent  and a luminometer  as described elsewhere To obtain heterotrophic plate counts (HPC), samples were serially diluted in sterile distilled water and then inoculated onto nutrient yeast agar plates using the spread plate technique. All plates were incubated in dark at 22°C or 36°C for 3 and 7 days, respectively. Results were expressed as colony forming units (CFU) per ml of water sample.http://www.physics.csbsju.edu/stats/.Statistical data evaluation was performed with the MS Excel Data Analysis tool . The reproducibility for indirect/calculated data  was calculated by a propagation-of-uncertainty method. FCM data was not always measured in duplicate, due to practical constraints. In these cases, a 9% error (average coefficient of variation (CV) (n = 39)) was applied for representing FCM data. The residual chlorine concentration distribution box plot was created using on-line calculator on −1) pre-sampling flushing procedure. The latter differs from extreme flushing applied for network cleaning, which is operated with high velocities of 1.5–1.8 m s−1−1) does not cause any scouring actions. However, it should be considered that part of the samples, especially during the first minutes of the flushing, can cointain biofilm bacteria detatched in a result of pre-flushing Sample collection during this study elucidated some of the problems specific for this network and highlighted the broader importance of correct sampling procedures. Fire hydrants were selected as sampling points to enable direct access to the distribution network and avoid potential household effects −1) of the initial residual chlorine concentration (0.5 mg L−1) was left in the water sample. The data suggests that the residual chlorine in the network was not sufficient to inhibit microbial growth, concurring with earlier report from Prévost and colleagues −1−1 allows bacterial growth −1.In the introduction we proposed the straightforward hypothesis that biological parameters would show an increase between the point of treatment and a point during distribution in case of biological instability . Before Staining of bacteria with fluorescent dyes was previously suggested as a way to distinguish between viable and damaged bacteria in real water samples 3 to 6.3×104 cells mL−1 (mean = 1.6×104±1.2×104 cells mL−1 in average). In turn, the values from the distribution network point (n = 23) were significantly higher: intact cell concentrations ranged from 1.37×105 to 4.66×105 cells mL−1 (mean = 2.5×105±9.9×104 cells mL−1), and the ATP concentrations from 0.021 to 0.063 nM (mean = 0.038±0.012 nM). Moreover, a distinct pattern was apparent in the distribution network data, with values peaking at about 05:00–07:00 and again at 12:00–13:00. During both these events, the intracellular ATP data followed a similar pattern as the intact cell concentration data, with a good overall correlation . Although it is not evident exactly why the bacterial concentrations peaked at these specific time periods, a plausible explanation is a change in the flow velocity due to diurnal changes in water consumption by both industrial and domestic consumers. It was previously shown in laboratory scale experiments that increased flow velocity could lead to increased bacterial detachment from biofilms and a re-suspension of loose deposits, thus leading to an increase in suspended cell concentrations High frequency monitoring of a single sampling point revealed temporal instability in the distribution network. We monitored the effluent of one treatment plant and one point in the network with 1-hour intervals during a day (ca. 21 h). The sampling was arranged in such a way that the network sampling started 15 hours after the treatment plant sampling, which corresponded with the estimated water residence time (WRT) for this location. Detailed data sets of diurnal changes in the microbial quality of water mains, such as 5–5.63×105 total cells mL−1 and between 9.7×103–2.13×104 intact cells mL−1 (hence 2–5% intact cells) depending on WTP. The data confirms effective final disinfection in all treatment plants. The total cell concentration values of the drinking water samples from the distribution network (n = 49) varied from 1.62×105 cells mL−1 to 1.07×106 cells mL−1 and the range of the intact cell concentration was from 5.28×103 cells mL−1 to 4.66×105 cells mL−1 (3–59% intact cells). Notably, 50% of all samples contained more than 1.06×105 intact cells mL−1 corresponding to an increase of at least one order of magnitude in those samples compared to effluent water, which clearly shows that bacterial growth in the distribution network was not an isolated occurrence. The observed increase in intact cell concentration is likely related to the presence of assimilable organic carbon (AOC) in the distributed water. While AOC was not measured in the present study, previous data for two of the treatment plants were high  Full-scale distribution networks are complicated systems, not restricted to a single source or a straight distribution line 4 cells mL−1) are marked with green bullets. Yellow and orange colored bullets indicate higher concentrations, while the points with the highest values (over 2×105 cells mL−1) are shown as red bullets. As could be expected, the map shows that the points with the lowest cell concentrations are mostly concentrated in areas close to the water treatment plants. Low intact cell concentrations in those areas could be ascribed to (1) disinfection during treatment and (2) growth inhibition from sufficient residual chlorine. Also the flow rate in the outgoing pipes closest to the treatment plants is high, which likely prevents water stagnation, sedimentation and cell adhesion on the pipe surface, and, consequently, biofilm formation and further bacterial growth. A different situation is observed in the distant areas from the water treatment plants and particularly in the so-called mixing zones, where the water from three different water treatment plants potentially mix. The map displays different color points spread in these zones without any visible order. The prevalence of the samples with higher cell concentrations there compared to the areas close to WTPs also corroborates the argument that increasing distance and water residence time could lead to chlorine decay with concomitant oxidation of dissolved organic matter; both these events would favor bacterial growth. Moreover, mixing zones are potential hot-spots for bacterial growth, as one water might well contain the nutrients that are growth limiting in the other.To examine the spatial distribution of the growth/instability in the network, the data was divided into four broad categories based on the extent of growth . These w4, 1.87×105, 2.51×105 intact cells mL−1). Such different intact cell concentrations could be due to several reasons: the time the samples were taken, which is linked to water consumption and the potential impact of which is shown in The uneven spatial distribution of the samples with different intact cell concentrations is noteworthy, highlighted for example by the three points in upper-left corner of the map. Based on the long distance from the WTPs, high intact cell concentrations were expected, but the samples taken from the hydrants located in this small area rather show variability  between intracellular ATP and FCM intact cell concentration is shown in −10 nM cell−1 ( = 3×10−17 g cell−1) with the average value of 1.68×10−10 nM cell−1 ( = 8.52×10−18 g cell−1) . The result is in the same range as ATP-per-cell values obtained from various water sources, which were analyzed with the same methods FCM and ATP data showed clear correlations, but these data did not correlate well with conventional HPC data. A total amount of 49 different samples was measured in duplicate with ATP  and FCM  analyses, while 38 of those samples were further analyzed with HPC. The significant linear correlation  was observed between HPC (at 22°C) and intact cell concentrations . Various studies were performed to compare ATP and HPC parameters from water samples, but good correlations were never observed e.g., R2 = 0.20 2 = 0.36 2 = 0.31 The conventional HPC results were compared with the FCM intact cell concentration values. A weak correlation , whereas HPC showed poor correlations with both parameters (R2 = 0.18 with intact cell concentration and R2 = 0.11 with intracellular ATP).A good correlation was observed between intracellular ATP and intact cell counts . While an improvement of the chlorination procedure could be a solution, the data suggests looking beyond only disinfection for achieving biological stability of drinking water.Figure S1Additional examples of hydrant flushing. Changes in intact cell concentration and intracellular ATP during flushing in 6 newly-opened fire hydrants. Intact cell concentration values are shown as solid lines with blue markers, whereas intracellular ATP results displayed as single green bullets.(TIF)Click here for additional data file.Figure S2Actual data for . Changes in various bacterial parameters between one water treatment plant and a randomly selected point in the distribution network Click here for additional data file.Figure S3Residual chlorine concentration in the distribution network. 50% of residual chlorine concentration in the network was between 0.12 (first quartile) and 0.23 (third quartile) mg mL−1, with a mean value of 0.17 mg mL−1 (n = 27). The whiskers indicate on minimum and maximum values, whereas bullets show outliers of the population.(TIF)Click here for additional data file.Table S1Physical and chemical parameters of water measured on-line during low velocity flushing of newly-opened fire hydrant. Some measurements were omitted during the first 20 minutes of flushing due to the high fluctuation in measuring tools readings.(DOC)Click here for additional data file.


Complex mixtures of molecular species, such as glycoproteins and glycosaminoglycans, have important biological and therapeutic functions. Characterization of these mixtures with analytical chemistry measurements is an important step when developing generic drugs such as biosimilars. Recent developments have focused on analytical methods and statistical approaches to test similarity between mixtures. The question of how much uncertainty on mixture composition is reduced by combining several measurements still remains mostly unexplored. Mathematical frameworks to combine measurements, estimate mixture properties, and quantify remaining uncertainty, i.e. a characterization extent, are introduced here. Constrained optimization and mathematical modeling are applied to a set of twenty-three experimental measurements on heparan sulfate, a mixture of linear chains of disaccharides having different levels of sulfation. While this mixture has potentially over two million molecular species, mathematical modeling and the small set of measurements establish the existence of nonhomogeneity of sulfate level along chains and the presence of abundant sulfate repeats. Constrained optimization yields not only estimations of sulfate repeats and sulfate level at each position in the chains but also bounds on these levels, thereby estimating the extent of characterization of the sulfation pattern which is achieved by the set of measurements. Complex mixtures of molecular species are common in biology; examples are antibodies4Many quantitative measurements on a mixture can be interpreted as weighted sums of species abundances, i.e. linear constraints on species abundances. Because relative abundances of species are between 0 and 1, a set of measurements corresponds to a bounded convex polyhedral setThe mixture studied in this paper is Heparan Sulfate (HS). HS consists of oriented linear chains of disaccharides having different levels of sulfation. It is ubiquitously found in mammalian tissues, mediates interaction between cells and extracellular matrix and has diverse biological functions in normal and pathological conditions121314151819p =  the vector of relative abundances of all possible n individual molecular species in a mixture. Values of pi are nonnegative and they sum to 1. One measurement b on the mixture yields a linear constraint: b is the overall proportion of a particular glycan in a glycoprotein mixture, then ai is the number of this glycan in glycoprotein form i. Uncertainty on measured values can be represented with inequalities and after adding slack and surplus variables to p this translates back into equality form. A vector b of m measurements is formalized as a set of linear constraints: Ap = b with p ≥ 0 and where A is a matrix. The polyhedral set pi is likely to require at least n measurements, even a small number m of constraints can greatly restrict some individual abundances. Consider indeed the following linear program:Call α = 1) and α = −1) correspond to smallest and largest possible values of pi. Quantity i, depends not only on types of measurements (matrix A) but also on their values (vector b) and thus can be less than 1 even when m < n. Two more general constrained-optimization frameworks are considered in this paper. In the first one, the optimization variable is vector p of individual species abundances:Solutions f is either a different linear function to estimate bounds on some mixture properties or the negative entropy to obtain maximum-entropy estimates. The second framework is utilized to investigate some structural features of the mixture by testing whether a model q(θ) of p can reproduce experimental data b or not:but objective function f is nonnegative and only zero for perfect fit. Obtaining f requires expressing some experimental measurements as explicit functions of model q(θ) and a methodology based on probability calculus is presented in this paper. Explicit constraints in optimization framework (3) depend on model type q and on some of the experimental measurements.where objective function d1... di... dn [reducing end], where di stands for disaccharide at position i. NMR measurements showed that the average chain length in BKHS is n = 16 disaccharides. BKHS chains can be cleaved by enzymes called heparinases. Utilizing a cocktail of heparinases I, III and IV, BKHS was fully cleaved, resulting fragments were separated and their relative abundances quantified, at the exception of fragments containing a non-reducing end. This gave 13 building blocks which were partitioned into two groups based on their 2-O-sulfation status: S for 2- O-sulfated and U for unsulfated. Indeed, 2-O-sulfation determines the propensity of cleavage by heparinase I and by heparinase III. Cleavage yields of U and S by these two enzymes were estimated with additional experiments and results are summarized in heparinase I or heparinase III were separated by their length and relative abundances of fragments which do not include a non-reducing end were quantified. Estimated fragment length distributions are presented in Modeling in optimization framework (3) is convenient for the mixture studied here, because Bovine Kidney HS (BKHS) consists of oriented linear chains of disaccharides: [non-reducing end] n = 16 the mixture still represents at least 216 = 65,536 different sequences. Yet, utilizing optimization framework (3) shows that the few measurements presented in Even after grouping disaccharides into S/U categories, with average chain length heparinases I and III, distributions of fragment length in heparinase digests reflect the pattern of sulfation along chains. To characterize this pattern, models of individual species abundances which allow for certain types of patterns are tested for their ability to reproduce heparinase digest data while preserving overall disaccharide composition. Pattern types are defined by two properties and their opposite. Nonhomogeneity (N) means that average sulfate level can vary with position in chains and homogeneity (H) refers to the opposite. Correlation (C) means that sulfation tends to occur in blocks along chains while independence (I) refers to lack of such correlation. There are four possible pattern types: H&I, H&C, N&I and N&C. It is shown by elimination that only combination N&C can explain experimental data. To facilitate presentation, results are shown here with models in which all BKHS chains have same length n = 16. In the n and some results are generalized to the case of a mixture of BKHS chains having different lengths.Because disaccharide categories S and U have different propensities of cleavage by s = d1... dn is the relative abundance ps of this sequence. Under H&I one obtains:A convenient approach to study a model of species abundances is to consider randomly drawing one chain from the mixture: the probability of drawing a sequence ρ (d) is the overall proportion of disaccharide d between positions i = 2 and n (γ of S and U at the non-reducing end (i = 1) do not contribute to experimental measurements but are still represented for completeness. Combining random drawing of one chain with its random cleavage at each disaccharide d based on heparinase yields c(d) (g(l) of fragment length l which is expected under H&I:where  2 and n . Proportlds c(d)  and utilc can be directly estimated from g (l) is bound to be an almost linear function of l. This is illustrated with n = 16. Modeled log-abundances show close to linear variations with fragment length l. Linear behavior is however not observed with experimental data (crosses). As shown in the n. Therefore, properties H&I cannot explain the experimental data. Nonhomogeneity or correlation must be incorporated.Parameter b at position i + 1 can depend on disaccharide a at position i but not on the value of i. Such a model allows for overrepresentation of species having blocks of sulfate. Model parameters are transition probabilities Pab which satisfy three types of constraints:Correlation is now introduced in the form of a homogeneous Markov modelρ(a) is again the overall proportion of disaccharide a. The last constraint is known as balance equation and, combined with composition ρ at position i = 2, it guarantees that modeled abundances preserve overall disaccharide composition ρ. Under this H&C model, the relative abundance of species s = d1... dn is given bywhere heparinase and utilizing probability calculus gives the following expression for distribution g(l) of fragment length l in heparinase digest:Combining random drawing of one sequence based on the above abundances to random cleavage of this chain by a where l) and gz(l) the experimentally measured and modeled fragment length distributions after digestion by heparinase z. For each heparinase a maximum fragment length lz is defined (11 and 6 for heparinases I and III) and fz(lz) and gz(lz) stand for abundances of fragment of length at least lz. Then, solving the optimization problemMatrices Πheparinase digest data. Because the objective function might not be convex, simulated annealingP, constraints are enforced by projectionheparinase I digest data even for different values of BKHS chain length n (Pab for optimized H&C and for H&I (Pab = ρ(b)). Abundant repeats of S yield high proportion of fragments of length 1 in modeled heparinase I digest. This implies high abundance of long stretches of U along chains and thus relatively high abundance of long fragments in modeled heparinase I digest. But as shown by means finding a homogeneous Markov model which best fits length n . Model Hn × 2 matrix Γ, where Γij (Γi(d)) is the proportion of disaccharide j (d) at position i from the non-reducing end. Matrix Γ has the following constraints:Nonhomogeneity of sulfation along chains is now introduced in the form of a ρj is the overall proportion of disaccharide number j. The last constraint preserves overall disaccharide composition. Assuming independence between positions, the N&I model yields the following relative abundance of species s = d1... dn:where ρ when Γ satisfies constraints (10). Composition Γ1 at the non-reducing end does not intervene in calculations summarized next. Combining random drawing of one chain with heparinase and utilizing probability calculus yields distribution g(l) of fragment length l which is expected under N&I:These abundances preserve overall disaccharide composition α = (n − 1)c, c = ∑bc(b)ρ(b) and ci = ∑bc(b)Γi(b) [2 ≤ i ≤ n].with ω can be efficiently computed, the problem of optimizing Γ so as to fit experimentally observed distribution fz(l) of fragment length in heparinase z digest can be solved in reasonable time:Because quantities n  and running again simulated annealing yields final values of the objective function which are about three times larger  is utilized. Results are first presented with a mixture where all chains have same length b as linear constraints Ap = b on vector p of molecular species abundances. For overall proportion ρj of disaccharide j between positions 2 and n one hasThe first step consists in expressing experimental measurements r is the number of disaccharide j in molecular species s. Calling fz(l) the relative abundance of fragments of length l after digestion by heparinase z and d1... dn the sequence of species s, the resulting constraint iswhere q of Als is the expected number of fragments of length l when cleaving sequence s by heparinase z and the denominator is the expected number of fragments in the entire mixture. Expressions of q are as follows:Numerator heparinase digests provide fifteen more constraints. Compatibility between constraints is then tested by solving a phase-I linear problem with artificial variables x:After including a constraint for all abundances summing to 1, overall disaccharide composition provides one linearly independent constraint and n in heparinase digest and overall disaccharide composition constraints. Infeasibility becomes close to numerical precision when n ≥ 13. Since the experimentally determined average BKHS chain length is n = 16 disaccharides, one can state that experimental measurements are compatible with each other because there exist vectors p of species abundances which can explain all measurements. Solving a different feasibility problem provides another evidence that homogeneity of sulfation level along chains is not supported by heparinase digest data. Constraint (14) is replaced with constraints imposing same disaccharide composition at each position i from the non-reducing end:The final value of the objective function is called infeasibility and should be close to numerical precision if constraints are compatible with each other. Obtained values of infeasibility are presented as a function of BKHS chain length heparinase digests are displayed with crosses as a function of BKHS chain length n in n = 16, it never reaches numerical precision and thus provides another evidence for nonhomogeneity of sulfation level along chains.Estimated values of infeasibility with these constraints combined with those of p is highly underdetermined . A standard approach for underdetermined problems is maximum-entropy modelingp: p* has been estimated, resulting mixture properties are examined. Circles in p*. Comparing maximum-entropy values to overall disaccharide composition  suggests that sulfated disaccharides S are more likely to be abundant near the non-reducing end (2 ≤ i ≤ 6). High abundance of S is also possible at the reducing end (i = 16). It is informative to compare relative abundances of BKHS sequences obtained under p* to abundances estimated with a less constrained maximum-entropy model based only on overall disaccharide composition, i.e. model H&I. In both models the most abundant species is xU15, where x stands for the disaccharide at the non-reducing end. However, abundances of species containing S near the non-reducing end or repeats of S tend to be higher with p* than with H&I. For instance, species xS4U11 is about 139 times more abundant under p* than under H&I. Further characterization of model p* is provided in the With only constraints (14) and (15) the problem of estimating p* of the polyhedral set i from the non-reducing end, the objective function in optimization framework (2) is now set to the following linear function: f (p) = αcTp with α = ±1 and cs = 1 if species s has sulfated disaccharide S at position i and 0 otherwise. Solution to this problem for α = 1 yields the lower bound for proportion of S at position i and setting α = −1 gives the upper bound. Estimated bounds are displayed with black squares in i = 1 is not restricted. For i ≥ 2, ranges of allowed proportions of sulfated disaccharide S vary with i. Consistent with maximum-entropy results, higher proportions of S are allowed towards the non-reducing end (2 ≤ i ≤ 6), while U must represent at least 50% of the disaccharides near the reducing end (7 ≤ i ≤ 14). Another noticeable result displayed in i = 16). This is lower than the 75% required by model N&I to best fit heparinase digest data  suggests that disaccharides S are likely to be less abundant near the reducing end but, when present there, might display block structures.Patterns of nonhomogeneity are rather opposite between model N&I  and maxip* ; the latn. To check that this does not induce nonhomogeneity and correlation, results are verified with models where chain length has nonzero variance. Call n =  a vector of increasing chain lengths and w the vector of their relative abundances. Distribution w has the following constraints: w ≥ 0, 1Tw = 1 and nTw = 16, the last one imposing an average chain length of 16 disaccharides. Simple models of w are obtained by first setting w(n) to the integral of a Gaussian density of mean 16 and standard deviation σ between lengths n and n + 1, and then projecting onto the polyhedral set defined by distribution constraints. Panel (a) of w for σ = 1.5 and σ = 3.5, when n− = 10 and n+ = 20.Results presented so far were with a model in which all BKHS chains have same length w translates into linear constraints Ap = b on vector p of species abundances: bn = w(n) and Anj = 1 if species j has length n and 0 otherwise. After adding constraints for overall disaccharide composition and heparinase digest data, optimization framework (2) is utilized to examine properties of the mixture. Panel (b) of p*: short chains have higher overall proportion of S. A relevant question is then whether variation of S with chain length is implied by heparinase digest constraints or rather just possible and occurring under maximum-entropy modeling. Adding constraints of same overall disaccharide composition for all chain lengths and solving the resulting feasibility problem (17) yields that there exist feasible vectors p. Relationship between chain length and disaccharide composition is therefore information which was not provided by heparinase digests. In other words, utilizing optimization framework (2) suggested that characterization of BKHS size fractions is an informative complementary approach.The set of all molecular species is now the set of all S/U sequences of length between 10 and 20. Distribution σ = 3.5, n− = 10 and n+ = 20 are summarized in n = 16: larger proportions of S are allowed towards the non-reducing end. Note that estimated bounds near position 20 are 0 and 1 when chains are aligned by their non-reducing end. This can be achieved while preserving all constraints because only a subset of chains have length 20. Bounds near the reducing end are better estimated after aligning by reducing end, because this estimation includes all chains. Result is then that proportion of S is more limited near the reducing end. Panels  of Results obtained with optimization framework (2) for In summary, utilizing optimization framework (2) provides insight into the potential structure of BKHS with only seventeen linearly independent constraints even though the last considered BKHS mixture model represents more than two million individual species. Optimization framework (2) suggests nonhomogeneity in the form of higher sulfation levels towards non-reducing end and higher correlation of sulfation state between adjacent disaccharides near reducing end. Nonhomogeneity and correlation might reflect different aspects of sulfation mechanisms which take place during BKHS biosynthesis.heparinase digests. One potential limitation of optimization framework (2) is a very large number of molecular species. Because constraint matrices are sparse, memory usage and computation time can be reduced by implementing special linear algebra and optimization methods31O-sulfation but also N-sulfation, 3- O-sulfation and 6- O-sulfation results in sixteen possible states for each disaccharide and the number (16)n of species becomes prohibitive for utilizing optimization framework (2). One would then have to explore the possibility of working with a parametric model, i.e. optimization framework (3) with a nonhomogeneous Markov model having 256 × n parameters. More generally, when the number of species is too large, a modeling choice such as partial independence might be required. Consider for instance that individual species are defined by sequences of three attributes: abc. If overall composition of c is known, assuming independence of c with other attributes yields p(aibjck) = p(aibj)p(ck). The mixture is then represented by all ab sequences, a vector of smaller dimension. Contribution h(aibj) of sequence aibj to a constraint is given by ∑kh(aibjck)p(ck), which can be estimated via sequence enumeration.Applying constrained-optimization framework (2) to BKHS experimental measurements showed that combining a few selected measurements can provide deep insight into the structure of a mixture having potentially millions of molecular species. Maximum-entropy modeling was the obvious first approach to model a mixture with only a few measurements. Yet, linear programming provided a more critical view of mixture properties. First, it demonstrated inability of homogeneity to explain experimental data. Second, it provided bounds on nonhomogeneity along chains, thereby estimating the extent of characterization achieved by the set of measurements with respect to this feature. Intervals provided by bounds account for remaining uncertainty and thus would be appropriate, for instance, to compare the sulfation pattern along HS chains in two different tissues. Non-overlapping intervals would hint at dissimilarity while overlapping intervals would not preclude similarity. Third and finally, linear programming showed that characterizing BKHS size fractions yields information complementary to heparinase digest as an explicit function of parameters of a species abundance model. Calculation methods were briefly mentioned and are detailed in the l. These equations would explain how nonhomogeneity along BKHS chains translate into potential variation of disaccharide composition with fragment length l. Such a mathematical exercise explains how properties of cleavage fragments, which are in general easier to experimentally characterize, reflect features of uncleaved chains. Moreover, as was illustrated with model H&I, some equations can be analyzed independently of parameter values and can then yield strong statements.Optimization framework (3) was utilized to demonstrate that only combination of nonhomogeneity and correlation could reproduce experimental data. This required deriving distribution of fragment length in a In conclusion, constrained optimization and mathematical modeling can yield deep insight into the structure of complex biochemical mixtures when applied to the combination of a small number of measurements. Careful selection of measurement types is crucial to provide complementary views of a complex mixture4How to cite this article: Pradines, J. R. et al. Combining measurements to estimate properties and characterization extent of complex biochemical mixtures; applications to Heparan Sulfate. Sci. Rep.6, 24829; doi: 10.1038/srep24829 (2016).


The fungal cell wall forms a compact network whose integrity is essential for cell morphology and viability. Thus, fungal cells have evolved mechanisms to elicit adequate adaptive responses when cell wall integrity (CWI) is compromised. Functional genomic approaches provide a unique opportunity to globally characterize these adaptive mechanisms. To provide a global perspective on these CWI regulatory mechanisms, we developed chemical-genomic profiling of haploid mutant budding yeast cells to systematically identify in parallel those genes required to cope with stresses interfering the cell wall by different modes of action: β-1,3 glucanase and chitinase activities (zymolyase), inhibition of β-1,3 glucan synthase (caspofungin) and binding to chitin (Congo red).wsc1Δ, elo2Δ and elo3Δ, showed a significant decrease in β-1,3-glucan synthase activity.Measurement of the relative fitness of the whole collection of 4786 haploid budding yeast knock-out mutants identified 222 mutants hypersensitive to caspofungin, 154 mutants hypersensitive to zymolyase, and 446 mutants hypersensitive to Congo red. Functional profiling uncovered both common and specific requirements to cope with different cell wall damages. We identified a cluster of 43 genes highly important for the integrity of the cell wall as the common “signature of cell wall maintenance (CWM)”. This cluster was enriched in genes related to vesicular trafficking and transport, cell wall remodeling and morphogenesis, transcription and chromatin remodeling, signal transduction and RNA metabolism. Although the CWI pathway is the main MAPK pathway regulating cell wall integrity, the collaboration with other signal transduction pathways like the HOG pathway and the invasive growth pathway is also required to cope with the cell wall damage depending on the nature of the stress. Finally, 25 mutant strains showed enhanced caspofungin resistance, including 13 that had not been previously identified. Only three of them, This work provides a global perspective about the mechanisms involved in cell wall stress adaptive responses and the cellular functions required for cell wall integrity. The results may be useful to uncover new potential antifungal targets and develop efficient antifungal strategies by combination of two drugs, one targeting the cell wall and the other interfering with the adaptive mechanisms.The online version of this article (doi:10.1186/s12864-015-1879-4) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae is formed by an inner layer containing β-1,3 glucan, β-1,6 glucan and chitin, and an outer layer composed of mannoproteins glucose (4 × 104 cpm/200 nmoles), 150 μM GTPγS and membrane enzyme (20-40 μg of protein) in a total volume of 40 μl. All GS assays were carried out in duplicate and data for each strain were calculated from at least three independent cell cultures. Statistical data analysis were done using the GraphPad V5 software.Cell extracts and GS assays were performed essentially as described previously . MembranSaccharomyces Genome Database (SGD). Statistically significant functional groups, appearing in our lists at a frequency greater than it would be expected by chance, were obtained by means of Gene Ontology (GO) tool “GO Term Finder” (version 0.83) using default settings at the SGD link with GO category “Process”, p-value ≤ 0.01 and False Discovery rate (FDR) option. The Heat map shown in Fig. Genes were assigned to functional categories using the yeast Gene Ontology (GO) tool “GO Slim Mapper” and further manually refined according to annotations of the BIOBASE Knowledge Library Proteome and the Not applicableOther information is provided as supplementary files.


It is commonly assumed that neural systems efficiently process natural sensory input. However, the mechanisms by which such efficient processing is achieved, and the consequences for perception and behaviour remain poorly understood. Here we show that small conductance calcium-activated potassium (SK) channels enable efficient neural processing and perception of natural stimuli. Specifically, these channels allow for the high-pass filtering of sensory input, thereby removing temporal correlations or, equivalently, whitening frequency response power. Varying the degree of adaptation through pharmacological manipulation of SK channels reduced efficiency of coding of natural stimuli, which in turn gave rise to predictable changes in behavioural responses that were no longer matched to natural stimulus statistics. Our results thus demonstrate a novel mechanism by which the nervous system can implement efficient processing and perception of natural sensory input that is likely to be shared across systems and species. The neural mechanisms underlying efficient coding of natural sensory stimuli have yet to be fully determined. Here, monitoring sensory pyramidal cells in weakly electric fish, the authors find SK channels are necessary for matching the responsiveness of neurons to natural stimuli and subsequent behavioural responses. Understanding the key computations by which neurons process incoming natural sensory stimuli, thereby giving rise to perception and behaviour, remains a central problem in neuroscience. There is growing evidence that sensory systems developed coding strategies to suit a dynamic range of statistics in natural sensory stimuli12346710Gymnotiform wave-type weakly electric fish sense amplitude modulations (AM) of their self-generated quasi-sinusoidal electric organ discharge (EOD) through peripheral electroreceptors found on their skin. These electroreceptors in turn send afferents onto sensory pyramidal neurons within the electrosensory lateral line lobe (ELL) that subsequently project to higher brain areas, thereby mediating perception and behavioural responses13151515151718The responses of electrosensory neurons to first-order electrosensory stimulus attributes have been well characterized  channels mediate adaptation to envelopes in pyramidal neurons. Indeed, both pharmacological activation and inactivation of these channels altered the degree of fractional differentiation and tuning to envelope stimuli, thereby reducing efficiency of processing of natural stimuli. Importantly, these manipulations caused predictable changes in behavioural responses to natural stimuli by inducing a mismatch between behavioural sensitivity and natural stimulus statistics. Our results therefore reveal a general mechanism by which SK channels can enable efficient processing and perception of natural stimuli through scale-invariant adaptation.n=14) in awake and behaving animals  to sinusoidal envelope stimuli with frequencies spanning the behaviourally relevant range (0.05–1 Hz). We found that pyramidal neurons responded to such stimuli through sinusoidal modulations in firing rate that increased in amplitude as a function of frequency (2πf)α and phase απ/2 (see Methods), where f is the frequency and α is the order of differentiation. We thus fitted a fractional derivative model with α=0.4 to our data (see Methods) and found an excellent fit  as a function of the adaptation strength. Indeed, both lower and higher adaptation strength led to tuning curves that were not matched to natural stimulus statistics and lowered coding efficiency as quantified by lower white index values . The second is that temporal whitening is only achieved for a given adaptation strength. Thus, increases or decreases in the adaptation strength will alter neural tuning and lead to sub-optimal processing of natural stimuli.To test whether pyramidal neurons display scale-invariant adaptation, we recorded their responses to step changes in envelope . We foun6To test our hypothesis, we plotted the time-dependent firing rate in response to steps with different durations. The curves obtained did not overlap and were each well fit by exponentials but with different time constants . Rescaliαneuron that is precisely related to the power law exponent of the stimulus αstim. Our model predicted that such fractional differentiation can be explained by including an adaptation current whose timecourse follows a power law which was confirmed experimentally. Importantly, our model also predicted that changing the level of adaptation can strongly affect αneuron, which should decrease coding efficiency. Thus, we next tested experimentally whether modifying adaptation in pyramidal neurons will alter their tuning exponent αneuron, and whether this will decrease coding efficiency as quantified by the white index.So far, we have shown that ELL pyramidal neurons can efficiently process natural stimuli through temporal decorrelation because of fractional differentiation, which ensures that the neural tuning increases as a power law with exponent et al.4139We focused on small conductance calcium-activated potassium (SK) channels. This is because previous results have shown that pharmacologically activating and inactivating these currents will increase and decrease adaptation in ELL pyramidal neurons, respectively39αneuron as neural sensitivity should then increase less steeply as a function of frequency when using sinusoidal stimuli. In contrast, we expect that EBIO application will increase the neural tuning exponent αneuron as neural sensitivity should then increase more steeply as a function of frequency. Consistent with these predictions, neural sensitivity indeed became relatively independent of frequency following UCL application as quantified by a decrease in αneuron . To test our hypothesis, we injected UCL and EBIO bilaterally into the ELL  are not also processed efficiently. This is because previous studies have shown that both first- and second-order attributes are processed in parallel by different subset of neurons in higher order areas2223Our results show that efficient processing instead emerges at the level of the ELL and requires SK channels. It is important to note here that we only recorded from pyramidal cells within LS, which displays the greatest SK channel expression3027285539Thus, it is likely that electrosensory-coding strategies are constrained to efficiently process natural stimuli. However, these will differ depending on the subset of natural stimuli considered and are likely to involve multiple sensory modalities. A complete understanding of these will require further studies and is clearly beyond the scope of this paper that only considered second-order electrosensory stimulus attributes.It is very likely that our results will be applicable to other systems. First, we note that SK channels found in weakly electric fish display ∼86% sequence identity with those found in mammals5107838Apteronotus leptorhynchus was used exclusively in this study. Animals were purchased from tropical fish suppliers and were acclimated to laboratory conditions according to published guidelinesThe weakly electric fish −1 when placed in the recording tank. To stabilize the head during recording, a metal post was glued to the exposed area of the skull. A small hole of ∼2 mm2 was drilled over the caudal lobe of the cerebellum above the ELL in order to gain access to the pyramidal neurons.A total of 0.1–0.5 mg of tubocurarine (Sigma) was injected intramuscularly in order to immobilize the fish for experiments. The fish was respirated through a mouth tube at a flow rate of ∼10 ml minWe used well-established techniques to make extracellular recordings with Woods metal electrodes from pyramidal cells within the LS of the ELL2, 1 mM MgSO4, 1 mM NaHCO3 and 0.5 mM NaH2PO4. The pH of the saline solution was 6.8. Glutamate (Sigma), UCL-1684 Ditrifluoroacetate hydrate (Sigma) and 1-EBIO 1-Ethyl-2-benzimidazolinone (Sigma) were dissolved in saline for application as beforeThe composition of the vehicle/control saline is as follows : 111 mM NaCl, 2 mM KCl, 2 mM CaCl−1 with duration of 50 s were presented. Previous studies have shown that such stimuli will reliably elicit a jamming avoidance response and/or transient EOD frequency excursion  responses in A. leptorhynchusαbehaviour was obtained by fitting a power law to the behavioural sensitivity as a function of frequency.Animals were immobilized and set up in the recording tank similarly to the method described above. However, both ELLs were exposed and two glass micropipettes loaded with saline control solution, UCL-1684 (100 μM), or 1-EBIO (2.5 mM) solution were inserted into the LS segment using previously established techniques42A. leptorhynchus is neurogenic, and therefore is not affected by injection of curare. All stimuli consisted of AMs of the animal's own EOD and were produced by triggering at the zero crossing of each EOD cycle as done previouslyαstim=−0.8 and whose phase varied uniformly. The slope of the spike triggered average computed in response to the noisy AM waveform was used to assign each cell as either ON of OFF-type as done previouslyThe EOD of α/dtα, in which the order of differentiation, is a non-integer number. In the frequency domain, fractional differentiation of order α corresponds to filtering by a transfer function H(f) given byFractional differentiationG(f) and phase φ(f) of the model can then be written asThe gain Im[H(f)] and Re[H(f)] are the imaginary and real parts, respectively. We fitted a fractional differentiation model to our data using the Grunwald–Letnikov definition, which was adapted to use a vectorization method to pass signals through a spectrum of fractional derivative values between 0 and 1 from which we obtained αneuronwhere Prr(f) is related to the gain G(f) and the stimulus power spectrum Pss(f) by the following equation:Linear response theoryαstim and if the neural gain increases as a power law with exponent αneuron, then we haveThus, if the stimulus power spectrum decays as a power law with exponent f if 2αneuron+αstim=0 or, equivalently, ifThe response power spectrum will then be independent of frequency αneuron and the behavioural exponent αbehavioural are related byWe assume that the neural tuning exponent We then haveα is the change in exponent α resulting from pharmacological manipulation of SK channels.where ΔTo model the responses of the pyramidal neurons to the stimuli used in this study, we implemented a leaky integrate-and-fire model with power-law adaptationC is the membrane capacitance, gleak is the leak conductance, Eleak is the leak reversal potential, I is a constant bias current, ξ(t) is gaussian white noise with zero mean and s.d. unity, σnoise is the noise intensity, s(t) is the stimulus which was taken to have the same statistics as for the data, σstim is the stimulus intensity, V is the membrane potential and z1(t) is the adaptation current. Each time the membrane potential reaches the threshold θ, it is reset to Vreset and an action potential is said to have occurred at that time. The adaptation current is then incremented.where N variables z1…zN that obeyed the following system of differential equationsWe approximated the power law adaptation using tj are the spikes times, δ(t) is the delta function, and b and γ are constants that determine the strength and power law exponent αneuron of the neural sensitivity, respectively. The model was simulated using an Euler–Maruyama integration with timestep dt=0.025 ms. We used parameter values C=1 μF cm−2, gleak=0.36 μS cm−2, Eleak=−70 mV, I= μA cm−2, σnoise= μA cm−2, N=40, b=0.2, γ=1.1253, θ=−50 mV, Vreset=−70 mV and C=1 μF cm−2. For these parameter values, we obtained αmodel=0.4.where stim−αbehaviour| and thus is maximum when the two power-law exponents match. This method was used in order to quantify the optimality of the animal's behaviour during the pharmacology experiments. For step envelope stimuli, we constructed PSTHs by averaging over each step onset and offset and typically used 50 bins for a given step duration.We used several methods in order to quantify our experimental data. Correlation time was measured as the duration of time it took to decay to 5% of maximum autocorrelation value. White index was measured by taking the normalized area under the power spectrum curve using a trapezoidal method and dividing by the maximum normalized area to achieve a value between 0 and 1. The match between behaviour and natural stimulus statistics was obtained as 1−|αHow to cite this article: Huang, C. G. et al. Temporal decorrelation by SK channels enables efficient neural coding and perception of natural stimuli. Nat. Commun. 7:11353 doi: 10.1038/ncomms11353 (2016).Supplementary Figures 1-3