This research investigated volatile profiles of over-ripe Thai mango purée during thermal processing by solid-phase extraction, volatile quantification by XAD-2-solvent extraction, as well as descriptive sensory analysis. Overripe fruits of three varieties were analyzed for the ripening stage using specific gravity as well as firmness and the physiochemical properties were also reported. We found that aromatic profiles could be used as true representative to describe Thai mango identities of each varieties. A simple and straightforward heat treatment had differing effects on aroma characteristics and those effects were dependent with mango varieties. Indeed, the amount of terpene hydrocarbons and oxygenated sesquiterpenoids alternated after heat treatment. All descriptive attributes of heated ‘sam-pee’ purée were intensified while, heat treatment significantly improved only “mango identity” in ‘maha-chanok’ and “fermented” odour in ‘keaw’ purée. With or without heat treatment, the volatile profiles of ‘maha-chanok’ remained quite stable while heating played a significant role on chemical ingredients of ‘keaw’ and ‘sam-pee’. Our study demonstrated that the manufacturing of the over-ripe mango into the products of high market value, selection of varieties is vitally important based upon their specific aroma characteristics before and after processing. Mango is one of the world’s most traded tropical fruits with a global annual production of approximately 50 million metric tons between 2016–2018 . In ThaiExcessive of the supply over the demand, may lead to a decrease in price and unsold produce. Therefore, over-ripe mangoes are usually shifted to food processing industries. Finished products include canned 49%) and dried (6%) mangoes which accounted for more than 50% of exported Thai mango products 9% and dr. During et al.  and their concentrations were designated in percentage.et al. [2 5 μm, 250×4.6 mm I.D column with acetonitrile:water (75:25) as mobile phase. The flow rate was 1 mL/min, and the column temperature was 40°C. Separated sugars were detected using RID detector . Fructose, glucose, and sucrose were identified by the retention of time and quantified using the standard curves of the individual sugars. Maltose at a concentration of 0.1 M was used as internal standard.For sugar contents, the extraction method of Wang et al.  was follThe above purée was then heated with constant stirring until the temperature reached 85°C for 15 min. After heating, the purée was cooled immediately to 35°C, placed in a plastic Ziploc bag, sealed, and kept at -18°C until used for analysis.et al. [Mango volatiles were analysed using automated Solid Phase Microextraction (SPME) after the methods of Beaulieu and Lea  and Wen et al.  with somTen accessors  age ranged from 21–40 years old evaluated the fresh and heated purée prepared from each of the three varieties. Training was intensively conducted in a 3 session per week for 4 weeks. The minimum of 6 descriptive attributes that best described mango aroma characteristics were generated in the discussion session by consulting with SPME volatile profiles analysed previously. Fresh and heated purée samples of each single variety (50 mL) were served in a 100 mL plastic cup with lids . The panoC. Prior to GC-MS [As described therein, the purée (100 g) was homogenised with 200 mL of chilled (4–8°C) phosphate buffer . Thereafter, phase separation was achieved by centrifugation at 5,000 rpm for 30 min (4–8°C). The supernatant was passed through a column (50 cm length x 1 cm i.d.) packed with 20 mL of AmberliteXAD-2 and method of Lalel et al. ,29 was fto GC-MS .http://www.heatmapper.ca). For the statistical analysis of the consumer test, the degree of liking data were subjected to the Analysis of Variance (ANOVA) followed by Duncan’s multiple range test at p < 0.05. Unless stated otherwise, the results are illustrated as mean ± SE (standard error) of triplicate analyses. The relationship between mango varieties (with heat treatment) and volatiles was analysed using principal component analysis (PCA). The volatile data were also subjected to agglomerative Hierarchical Clustering (AHC) for simply interpretation with a PREFMAP feature in the statistical package.All analyses were done using the XLSTAT version 2018. Scatter plot and box plot graphs were used to evaluate the correlation of specific gravity and mango variety. Exponential decay nonlinear regression analysis was fitted for accessing the relationship between the specific gravity and firmness at confidence level of 95%. Heat mapping of free organic volatiles was generated using an online software  and also polysaccharide degradation which led to breakdown of starch, cellulose and hemicellulose ,37. ThisFor this instance, we anticipate that the loss of firmness due to the conversion of insoluble pectin into soluble forms during the ripening stage additionally contribute to the increase in specific gravity particularly before the fruits reached the over-mature stage ,40,41. Bet al. [et al. [The physicochemical properties of the over-ripe mango pulp  of each variety were determined . From thet al. . The det [et al.  reportedet al. [TSS is one of the main parameters to describe maturity index as well as an indicator for mango fruit quality . Althouget al.  reportedet al. . In our et al. .Solid-phase microextraction (SPME) is a rapid, sensitive and non-solvent metabolomics tool for exclusive characteristics of the aromatic volatiles of fruit pulp as well as the whole fruit in food process industry ,28. The et al. [From the result of the organic volatiles, we then nominated 5 descriptors for the later sensory analysis viz., “citrus”, “green”, “mango identity”, “tropical fruit”, and “sweet and floral” . The sixet al.  advised Aroma perception is generally specified by a diverse range and intensity of volatile compounds synthesised through physiological changes of the fruits ,53. For For the calculation of odour activity value (OAV), odour thresholds of the volatiles from mango fruits of different varieties were used as reported by Pino and Mesa  along wiIn summary, heat processing altered the intensity of aromatic sensorial profiles of mango purée from 3 Thai varieties with high market need for processing. The analysis of free volatiles by GC-MS illustrated 19 volatile components leading with those representing mango identity such as δ-3-carene, β-mycene and limonene. The aroma profiles of Thai mango purée were also categorised based on the citrus and sweet-floral characteristics which were unique for Thai variety. The PCA with PREFMAP elucidated that the aromatic profiles could distinguish these varieties and heat treatment had largely influenced the profile in the purée of ‘keaw’, while ‘sam-pee’ and ‘maha-chanok’ could remain their aroma identity. The finding can be advantageous to fruit processing industry whose interest is to preserve the aroma identity of mango varieties through product and development process.S1 Dataset(PDF)Click here for additional data file.


Evaporative water loss during metabolic gas exchange is an unavoidable cost of living for terrestrial insects. This respiratory water cost of gas exchange (the ratio of respiratory water loss to metabolic rate) is suggested to depend on several factors, such as the mode of gas exchange (convection vs. diffusion), species habitat, body size and measurement conditions. We measured this cost for a blaberid cockroach and a tenebrionid beetle using flow-through respirometry. We controlled the factors that affect respiratory water cost of gas exchange, i.e., both species are similar in their mode of gas exchange (dominantly convective), habitat (relatively moist) and body size, and were measured at the same temperature. The cockroaches showed both continuous and discontinuous gas exchange patterns, which had a significantly different metabolic rate and respiratory water loss but the same respiratory water cost of gas exchange. The darkling beetles showed a continuous gas exchange pattern only, and their metabolic rate, respiratory water loss and respiratory water cost of gas exchange were equivalent to those cockroaches using continuous gas exchange. This finding from our study highlights that the respiratory water cost of gas exchange is similar between species, regardless of the gas exchange pattern used, when the confounding factors affecting this cost are controlled. However, the total evaporative water cost of gas exchange is much higher than the respiratory cost because cuticular water loss contributes considerably more to the overall evaporative water loss than respiratory water. We suggest that the total water cost of gas exchange is likely to be a more useful indicator of species distribution with respect to environmental aridity than just the respiratory water cost.Nauphoeta cinerea) and the darkling beetle (Zophobas morio), which are similar in their mode of gas exchange (dominantly convective), habitat (mesic), body size and measurement conditions, by measuring gas exchange patterns using flow-through respirometry. The speckled cockroaches showed both continuous and discontinuous gas exchange patterns, which had significantly a different metabolic rate and respiratory water loss but the same respiratory water cost of gas exchange. The darkling beetles showed continuous gas exchange pattern only, and their metabolic rate, respiratory water loss and respiratory cost of gas exchange were equivalent to those cockroaches using continuous gas exchange. This outcome from our study highlights that the respiratory water cost of gas exchange is similar between species, regardless of gas exchange pattern used, when the confounding factors affecting this cost are controlled. However, the total evaporative water cost of gas exchange is much higher than the respiratory cost because cuticular water loss contributes considerably more to the overall evaporative water loss than respiratory water. We suggest that the total water cost of gas exchange is likely to be a more useful index of environmental adaptation  than just the respiratory water cost.Respiratory water loss during metabolic gas exchange is an unavoidable cost of living for terrestrial insects. It has been suggested to depend on several factors, such as the mode of gas exchange (convective vs. diffusive), species habitat (aridity), body size and measurement conditions (temperature). We measured this cost in terms of respiratory water loss relative to metabolic rate (respiratory water cost of gas exchange; RWL/ This respiratory water loss (RWL) during breathing is considered a cost of gas exchange [Gas exchange by terrestrial insects takes place through the tracheal system. This system extends from spiracles on the exterior of the body, and inside the body as a network of air-filled tubules that branch further to become thin-walled tracheoles, which are in close proximity to tissues and are the site of gas exchange ,2. Spiraexchange .Water balance is a substantial problem for terrestrial insects due to their small body size and consequent large surface area to volume ratio. RWL, as a cost of gas exchange, varies depending on ambient temperature and environmental water availability, and is therefore typically a higher portion of total water loss for insects living in arid compared to mesic environments. RWL can be reduced by closing the spiracles, and the dynamics of their opening and closing are responsible for gas exchange patterns ,5. CuticInsects show three distinct patterns of gas exchange, based on their modulation of spiracle opening: Continuous, cyclic and discontinuous gas exchange  ,8. CGE a2 (PCO2) and O2 (PO2) between the ambient environment and the insect tracheal system. Spiracles remain closed during the C phase. The PO2 drops during the C phase due to metabolic consumption in tissues, whereas PCO2 rises as CO2 is produced and continues to dissolve in the haemolymph [PO2 [2 to maintain PO2 at a steady level (~2–4 kPa), whereas there is no CO2 loss against the bulk influx of O2, and PCO2 continues to rise [2 and release of CO2. The cycle starts again when intratracheal PCO2 reaches a threshold of ~4–6 kPa and the spiracles close [DGE, the most complex pattern of gas exchange, has three distinct phases of spiracular activity: Closed (C), flutter (F) and burst (B). These are produced by modulation of spiracle closure based on partial pressure differences of both COemolymph . The C pmph [PO2 . During  to rise . The B pes close ,15,16. Des close ,15.The mode of spiracular gas exchange (convective vs. diffusive) is likely to play an important role in determining the relationship between metabolism and RWL, i.e., the respiratory water cost of gas exchange RWL/V˙CO apart frcing RWL . For exacing RWL  notes thcing RWL ) found scing RWL . HoweverNauphoeta cinerea Olivier 1789; Blaberidae) and the darkling beetle . We expected similar respiratory water costs of gas exchange for both species, since both species use a same dominantly convective mode of gas exchange , arepumping, ,22,23, a2, 0.03% CO2) and ambient RH (range 60–80%).Speckled cockroaches were purchased from Livefoods Unlimited . They were maintained in a plastic container with a mesh lid, and the sides of the container were lined with Vaseline to prevent escape. Cockroaches were provided with dry cat food  and empty cardboard egg trays provided harbourage. Water-filled glass vials with cotton wool in the open end provided drinking water. Darkling beetles were purchased from Bugs ‘N’ Things . They were maintained in a plastic container covered with a cheese cloth lid. Bran and carrots were provided, along with drinking water. Cockroaches and beetles were maintained at a constant temperature of 25 °C ± 1 SE, 12 L:12 D photoperiod, ambient gaseous conditions  to minimise any effect of temperature variation on gas exchange patterns. Individuals were observed to be immobile (‘at rest’) during the respirometry. 2, and drierite  to absorb moisture. Some cockroaches were measured using air from a compressed air cylinder , which had low and constant background concentrations of CO2 and moisture and did not need to be scrubbed. The dry, CO2-free air flow entering the chamber was regulated at 25 mL min−1 STPD using a mass flow controller . Mass flow controllers were calibrated with a Gilian Gilibrator 2 . Excurrent air from the respirometry system entered a brass housing containing a temperature humidity probe  to measure relative humidity (RH) and temperature of excurrent air (Ta). The probe was calibrated using a DewPoint Generator DG-4  and a traceable mercury glass thermometer . The CO2 concentration of excurrent air was then measured with a CO2 analyser . The CO2 analyser was calibrated using a certified span gas (0.153 ± 0.003 (SE)% CO2, BOC Gases, Canning Vale, WA, Australia).Compressed air was passed through a column of sodasorb  to scrub CO2 analysers, respectively. These voltmeters were connected via a USB port hub  to a desktop PC. Voltage signals were sampled every 10 s and converted to ppm for CO2 concentration, percent relative humidity for water vapour and degree Celsius for temperature using a custom written program . Data were stored continuously in an Excel file during each respirometry trial. Acquired data were analysed with a laptop using an Excel spreadsheet (written by PC Withers and W Abbas). The raw values of CO2, H2O and temperature were first calibration corrected. The rate of CO2 emission (STPD −1 h−1) was calculated based on the following equations from Withers , V̇e is the excurrent flow rate of air (STPD mL min−1), FiCO2 is the incurrent fraction of CO2, FeCO2 is the excurrent fraction of CO2 and m is the body mass (g). All these variables were measured or calculated, except V̇e, which was calculated asiO2 is the incurrent fraction of O2, FeO2 is the excurrent fraction of O2, FiH2O is the incurrent fraction of H2O, FeH2O is the excurrent fraction of H2O and RER is the respiratory exchange ratio  were used to measure analogue signals from the Vaisala probes and COs .(1)V˙Cbe 0.85, ). The inquations .−1 h−1) was calculated asI is the incurrent flow rate (ATP mL min−1), V̇’e is the excurrent flow rate of air (ATP mL min−1) and WVD  is the water vapour density (ATP mg L−1), calculated from Ta and RH using hygrometric equations [The rate of evaporative water loss quations . The V˙C. . The V˙2 emission had a clear closed phase  were analysed for each insect to calculate −1 h−1), and the duration (sec) for each phase and the entire cycle (IB + B). DGE cycles were analysed for calculation of water loss rate (2 emission) as for The to zero) . For calto zero) ). DGE cy−1) and RWL (μg h−1) were calculated by the linear regression method [2O µL CO2−1), which is the ratio of RWL/ We partitioned evaporative water loss during the B phase of DGE into its cuticular (CWL) and respiratory (RWL) components after Hadley and Quinlan . This in method , where C−1 cm−2 hPa−1), the CWL (µg h−1) expressed per unit surface area  per saturation deficit  was calculated after Appel et al.,  asCP =The surface area of cockroaches was calculated using Meeh’s formula as cited by Gray and Chown  asSA =urements ,36. The urements ,37. For .  and Rol. . −1 using RER = 0.85) and RWL (−1) were regressed against each other.To compare the RWL cost of metabolic rate between patterns of two species, metabolic rate  was regressed against body mass  separately for CGE and DGE gas exchange patterns of cockroaches and of beetles. To fit the respiratory water cost of gas exchange for our species to the Woods and Smith [To compare the metabolic cost associated with gas exchange patterns, the metabolic rate and body mass of species of cockroaches (order: Blattodea) and of beetles (family: Tenebrionidae) were compiled from the published literature. For comparison, all metabolic rates were converted to 23 °C equivalence using a Q insects . The metnd Smith  model, and Smith  were usen). Physiological variables metabolic rate (−1 h−1), total evaporative water loss (−1 h−1), CWL (µg g−1 h−1), RWL (µg g−1 h−1), % CWL , % RWL  and CP (µg h−1 cm−2 hPa−1) of cockroaches showing CGE were compared with DGE cockroaches and with beetles showing CGE using general linear models in R  version 2.0 and α was 0.05 for all tests.Means are presented with standard error (s.e.m) and sample size . RWL ann = 15), with the reminder using CGE . None of the individuals switched between CGE and DGE during the 2-h respirometry duration. The cockroach DGE pattern clearly showed the three phases C, F  and B (2 was considerably higher during the B phase (90%) than IB . There was a clear correspondence of Of the 23 cockroaches measured, 15 used DGE  and B . Both IB IB 10%, . Beetlesinuously .−1 h−1) was 25% lower than CGE cockroaches (262 ± 33 µL g−1 h−1) and differed significantly . RWL, as a fraction of the total water loss, was significantly lower for cockroaches showing DGE than CGE , hence % CWL was significantly higher for cockroaches showing DGE than CGE . There were no significant differences for any other physiological variables between CGE- and DGE-breathing cockroaches : t28 = 0.52, p = 0.61; RWL (µg g−1 h−1): t28 = 1.53, p = 0.14; CWL (µg g−1 h−1): t28 = 0.26, p = 0.80; CP (µg h−1 cm−2 hPa−1): t28 = 0.69, p = 0.50; Within the same species, the metabolic rate of cockroaches showing DGE . Moreover, there were no significant differences in any physiological variables for cockroaches and beetles using CGE : t28 = 1.74, p = 0.09; RWL (µg g−1 h−1): t28 = 1.67, p = 0.11; CWL (µg g−1 h−1): t28 = 1.60, p = 0.12; CWL%: t28 = 0.06, p = 0.95; CP (µg h−1 cm−2 hPa−1): t28 = 1.58, p = 0.12; At the interspecific level, cockroaches using CGE (808 ± 54 mg) were 28% heavier than CGE-breathing beetles  and intercept . That is, there was no difference in metabolic rate for cockroaches using either CGE or DGE (p = 0.646) but a significantly different intercept (p = 0.001). In other words, beetles that used DGE had a lower metabolic rate than CGE beetles (Interspecific comparison of metabolic rate for CGE and DGE gas exchange patterns of cockroaches based on regression of logE or DGE a. Howeve beetles b.10RWL (mol H2O day−1) and log 2 day−1) of the individual speckled cockroaches using CGE or DGE and beetles using CGE resulted in a common slope , which indicates a constant respiratory water cost of gas exchange of about 0.76 mol H2O mol O2−1 (2O mol O2−1). The intercept was not significantly different between cockroach individuals using CGE or DGE (p = 0.374) and between cockroach and beetle individuals using CGE (p = 0.284). In other words, the respiratory water cost of gas exchange was similar within the same species for its individuals using different gas exchange patterns and between both species showing the same gas exchange pattern  and logO2 uptake (mol O2 day−1). The positions of CGE and DGE cockroaches and CGE beetles in the multispecies relationship suggest that both species conform to the proposed model for respiratory water cost of gas exchange (2O mol O2−1).The interspecific comparison of the respiratory water cost of gas exchange shows that DGE and CGE cockroaches and CGE beetles from our study fell with in the lower bound of the 95% prediction interval of Woods and Smith’s  multispeexchange b. There n = 15), with the remainder using CGE (n = 8). This is consistent with a previous study of this species [Aptera fusca) and Persiphaeria sp. [Eloedes obscura, Helea sp. and Brises blari [We found that speckled cockroaches maintained under the same housing conditions used either CGE or DGE patterns of gas exchange. Around two-thirds (65%) of cockroaches measured used DGE  is 56% higher than a previous measurement (125 µL g−1 h−1) for this species at same temperature [Tenebrio molitor) and lesser mealworms (Alphitobius diaperinus) [−1 h−1, adjusted to 23 °C assuming a Q10 value of 2, [−1 h−1). This was significantly higher than for DGE speckled cockroaches. The metabolic rate of DGE speckled cockroaches  . The metue of 2, ), is comZophobas morio conforms well to the relationship between body mass and metabolic rate for other tenebrionid beetle species using CGE  or DGE (0.635 mg h−1) are within the range (0.5–2 mg h−1) reported by Schimpf et al. [The total evaporative water loss rates of speckled cockroaches using CGE  used for flow-through respirometry with insects vary widely in their measurement range and precision. Due to their small size (body mass: 1 mg–4 g), smaller fluxes of CO2 from insects ideally require a higher measurement precision than measurement of larger animals  [2 or CO2 levels in the excurrent air) for small animals. Insect respirometry studies often use high-precision CO2 analysers  to characterise gas exchange patterns [Gas analysers   because patterns , but we patterns ,51, but patterns ,53,54. WThe metabolic rate and RWL, as a fraction of total evaporative water loss, differed for speckled cockroaches using CGE and DGE, whereas there was no significant difference between speckled cockroaches and darkling beetles using CGE . It is il., 2010 , for then = 3) using CGE are underrepresented compared to those using DGE (n = 8). Second, there might be phylogenetic nonindependence of species belonging to different families [0.94). The significant metabolic rate difference for beetle species between CGE and DGE might reflect the larger sample sizes for the beetle comparison, and all of the beetle species belong to the same family (Tenebrionidae), so the phylogenetic effect may be small. However, an important further caveat for interpretation of these interspecies patterns is that all species are not likely to have been acclimated to the same environmental conditions and food and water regimes and may differ in adaptation to their natural environments. It is likely that all of these possible differences (and more) influence metabolic rate, along with gas exchange pattern. Detailed studies of the plasticity of insect gas exchange patterns are rare apart from a single study on acute and chronic oxygen and moisture effect [At the interspecific level a, the mefamilies . In conte effect  and acute effect .2O mol O2−1) did not differ significantly for the speckled cockroaches using CGE and DGE, or the darkling beetles using CGE  and species is expected, so long as the diffusive or convective mechanisms for respiratory H2O and O2 exchange are the same. The values of both species for the respiratory water cost of gas exchange also conformed to the multispecies model of Woods and Smith [The respiratory water cost of gas exchange  based on the regression method is similar to the value from the direct method for the individuals showing DGE (4.48 ± 0.44 µg h−1 cm−2 hPa−1). The similar results from both these methods is consistent with an earlier study for a closely related blaberid cockroach, Perisphaeria sp., having CP calculated using both methods [Perisphaeria sp.  is a useful measure of water loss because it expresses the rate of cuticular water loss relative to the driving force for evaporation and body surface area. The CP for the speckled cockroaches using CGE , and th2 hPa−1, ). These These estimates for CP were determined using flow-through respirometry, and they differ from CP determined by gravimetric measurement. Gravimetric CP uses weight loss over time to calculate total water loss and is considerably higher for cockroaches and beetles ,60. The 2 during this phase. The speckled cockroach from our study releases 10% of its overall cycle Blatella germanica) about 6% [Perisphaeria sp. about 15% [Periplaneta Americana) about 27% [Schistocerca gregaria releases about 10% of its total Cataglyphis bicolor) releases about 14% of the total CO2 release [During the interburst phase of DGE, spiracles are generally considered to be completely closed. However, for some insects from various orders, there is a measurable release of COabout 6% , Perisphbout 15%  and the bout 27% . The desIB phase  and a de release .2 has not attracted much attention in the literature. There is no consensus on whether this CO2 release is spiracular [2, is a possible source of IB Calliphora vicina) while simultaneously measuring CO2 release. Alternatively, some studies [2 might diffuse across the cuticle  during the IB phase. Unequivocal universal support for either trans-spiracular or trans-cuticular release is lacking at present.The presence of this small , whereas beetle species using CGE had a higher metabolic rate than those using DGE . However, interspecies analyses such as this might confound other factors  with gas exchange pattern. The respiratory water cost of gas exchange did not differ between CGE and DGE, or speckled cockroaches and darkling beetles as we expected, but the overall water cost of gas exchange water loss for gas exchange was considerably higher, reflecting the substantial CWL compared to RWL. We suggest that overall water cost of gas exchange could provide a broader index of water economy that includes species differences in cuticular permeability, hence hygric adaptations to the species’ environmental aridity.


Nonradiative processes limit optoelectronic functionality of nanocrystals and curb their device performance. Nevertheless, the dynamic structural origins of nonradiative relaxations in such materials are not understood. Here, femtosecond electron diffraction measurements corroborated by atomistic simulations uncover transient lattice deformations accompanying radiationless electronic processes in colloidal semiconductor nanocrystals. Investigation of the excitation energy dependence in a core/shell system shows that hot carriers created by a photon energy considerably larger than the bandgap induce structural distortions at nanocrystal surfaces on few picosecond timescales associated with the localization of trapped holes. On the other hand, carriers created by a photon energy close to the bandgap of the core in the same system result in transient lattice heating that occurs on a much longer 200 picosecond timescale, dominated by an Auger heating mechanism. Elucidation of the structural deformations associated with the surface trapping of hot holes provides atomic-scale insights into the mechanisms deteriorating optoelectronic performance and a pathway towards minimizing these losses in nanocrystal devices. Charge trapping can lead to severe nonradiative losses in colloidal semiconductor nanocrystals (NCs). The authors report femtosecond electron diffraction measurements on photoexcited NCs to reveal atomic-scale insights into how localization of charges at trap sites induce surface deformations. Among these, colloidal semiconductor NCs have attracted significant technological interest due to their appealing optoelectronic properties4, which are tunable via shape, size, composition, and surface chemistry7. Today, state of the art NCs can reach near-unity radiative efficiencies8 but these are typically measured under moderately weak excitation conditions. In applications, such as lasers9, photodetectors10, multiexciton-harvesting solar cells11, and electrically pumped LEDs12, NCs are commonly exposed to high energy and/or high intensity excitation conditions, where nonradiative relaxation rapidly escalates.Nonradiative relaxation processes in materials represent fundamental loss mechanisms, which set performance limits in electronics, optoelectronics, and photocatalysis. Nonradiative relaxation events become further critical in devices of quantum-confined materials such as nanocrystals (NCs) and nanowires due to their high surface-to-volume ratios. As such, intensive research efforts have been focused on identifying nonradiative losses and the means to circumvent them in nanomaterials13, reduced photoluminescence quantum yields15, and increased photoionization16. These observations have suggested that hot carriers in NCs can lead to severe charge trapping, increasing nonradiative losses. In addition, Auger recombination becomes dominant in NCs that have more than one exciton17. A hot carrier is created at the expense of an annihilated exciton via an Auger process, hence substantially curbing the performance of NC lasers18 and LEDs19. Although earlier works focused on identifying optical signatures associated with such nonradiative processes in NCs21, more recent works have begun to point to the fundamental role of dynamic structural fluctuations interrelated with nonradiative relaxation in NCs29. In this context, neutron scattering measurements corroborated by molecular dynamics (MD) simulations22 and correlative transmission electron microscopy (TEM) studies27 have indicated that NC surfaces are mechanically soft, and thus may accelerate the nonradiative relaxation process. Nevertheless, such structural deformations associated with nonradiative relaxations in photoexcited NCs have remained elusive to date and have never been directly probed on ultrafast timescales.Previously, high photon energy excitation of NCs has been shown to cause increased blinking30 measurements on prototypical cadmium chalcogenide colloidal NCs to directly probe the atomic scale responses following photoexcitation. We investigate the effects of excitation photon energy on the transient atomic responses in thin film samples of core/shell and core-only NCs. Studying the core/shell sample under different excitation energies enables selective excitation of the core vs. the shell; thus, we decouple the effects of NC surfaces on nonradiative relaxations. We find that Auger recombination dominates the transient heating of the core/shell NCs when multiexcitons are generated in the core by photons with energies close to the bandgap of the core. The transient heating response is corroborated by MD simulations. On the other hand, we unveil that localized disordering is induced in addition to transient heating when multiexcitons are generated predominantly in the shell by photons with energies much larger than the bandgap of the shell. These localized structural deformations arise from localization of hot holes at NC surfaces forming surface small polarons , where Q is the scattering vector. The sample in Fig. I measured at a pump-probe delay of t ≈ 500 ps when the sample is excited at 510 nm. The intensity of all diffraction peaks, labeled from Q1 to Q7 /I0) represented at four different diffraction peaks at an excitation fluence of 2.1 mJ cm−2. Changes in I(t)/I0 become progressively larger for higher Q peaks. This Q-dependence resembles a transient heating response known as the Debye–Waller (DW) effect, where diffraction peak intensities decrease as the material heats up due to increased mean squared atomic displacements (〈Δu(t)2〉). In a DW model under harmonic assumption, I(t)/I0 can be related to 〈Δu(t)2〉 via 31. To check experimental agreement with the DW model, we plot −ln(I(t)/I0) as a function of Q2 in Fig. t = 1000 ps. A linear relationship with zero intercept holds for all fluences studied between 1.5 and 3.1 mJ cm−2 2〉 as shown in the inset of Fig. We first discuss measurements of the core/shell sample when excited at 510 nm. Note that 510 nm excitation predominantly excites the CdSe core  revealing transient changes in the atomic pair correlations33  resulting from a static temperature increase of ΔT  (T0 = 300 K) is plotted in Fig. G, further supporting our conclusion that the lattice response in this case is dominated by transient heating.To gain better insight into the structural deformations occurring in response to photoexcitation in these NCs, we calculate a differential atomic pair distribution function PDF) Δell Fig. c, inset ell Fig. . This inF Δell Fiu(t)2〉 into lattice temperature changes ΔT(t) by considering the DW factors calculated by our MD simulations  along with ΔE(t), which is the energy transferred to the NC lattice (Supplementary Section T reaches a quasi-equilibrium at ca. 13 K under an excitation fluence of 2.1 mJ cm−2 consistent with the MD simulation above. We fit ΔT(t) phenomenologically by a single exponential function, which gives lifetimes on the order of 200 ps  an Auger heating mechanism36. A bottleneck between hot carriers and optical phonons is not considered, as prior reports have established that this coupling is rather fast (≤1 ps) in colloidal NCs37. However, a bottleneck may exist in the down-conversion of the emitted optical phonons into acoustic ones, where the acoustic phonons are more prominent in the DW response because of their larger contribution to mean squared atomic displacements  as τAR is the biexciton Auger lifetime. τAR ≈ 625 ps in the core/shell NC /I0 measured at four different diffraction peaks, which shows that the transient structural responses occur significantly faster under this excitation (~20 ps). Figure I(t)/I0) over Q2 at t = 200 ps. Although low Q diffraction peaks (Q1–Q5) show a linear-like response among themselves  strongly deviate from this linearity. This deviation happens consistently for all studied fluences between 2.5 and 6.0 mJ cm−2  under an excitation fluence of 4.8 mJ cm−2. An investigation of the transient dips at different atomic correlations indicates that at early delay times the first nearest neighbor correlation at r = 2.5 Å is more affected than all other correlations  at different r. Supplementary Fig. r of 4.1 and 7.1 Å, which exhibit the same time constant of 20 ± 1 ps. Thus, the effect for large r correlations is the same and its response is dominated by the transient heating. On the other hand, ΔG at r = 2.5 Å exhibits considerably faster kinetics with a time constant of 11 ps. In this case, local disordering and transient heating both contribute to the dynamics together. To decouple the dynamics associated with the formation of localized deformations, Fig. G normalized by ΔG, where the normalization effectively removes the heating dynamics. After the normalization, we estimate a time constant of ~3.5 ps directly linked with the time scale for the formation of localized lattice deformations.To understand the nature of these localized structural deformations, we perform differential atomic PDF analysis. Figure I(t)/I0) measured at different Q peaks /I0 at lower Q peaks exhibit a time constant around ~23 ps dictated by the transient heating, the Q6 peak shows a time constant of ~10 ps. To extract the dynamics of the localized deformations in this case, we subtract I(t)/I0 measured at a low Q peak (Q3), scaled to the linear DW value at Q6, from that of the experimental data at Q6, obtaining a time constant of 6 ps   by a factor of 4 , which strongly implies that the transient structural response in the case of 340 nm excitation is dominated by the localized surface carrier trapping  Fig. , which iing Fig. .39. No significant localized deformations are observed with 510 nm excitation, which implies that the delocalized hot electrons cannot be the main cause of the surface trapping. Thus, hot holes must govern the formation of localized deformations as they trap at the NC surfaces. This is consistent with prior theoretical work in cadmium chalcogenide NCs, which have suggested that the main carrier that leads to trapping is the hole due to poorly passivated surface chalcogen atoms44. The spatial extent of the localized distortions arising from hot hole trapping can be estimated from the dynamic structural information (Supplementary Section Q6) reveal larger weighting of the l component of the (hkl) Miller indices /I0) as a function of Q2 for both 340 and 510 nm excitations. We observe that the localized lattice deformations, evidenced by an increased loss at high Q diffraction peaks, occur under both excitation cases, while the effect is much more pronounced for the 340 nm excitation. Differential atomic PDF analysis in Fig. We also measure the transient structural responses in a CdSe core-only NC sample, which is the same size core used in the core/shell sample. Figure Femtosecond electron diffraction applied to colloidal semiconductor NCs directly visualizes nonradiative relaxations occurring in photoexcited semiconductor NCs in real time with an atomic-scale resolution. With this, we uncover the dynamical structural responses associated with the formation of localized surface charge traps and Auger recombination. We show that hot holes with excess kinetic energy induce short range atomic deformations extending ~0.15 Å as these carriers localize at surface trapping sites and form surface small polarons. Our results indicate that excitation energy management in NCs by minimizing the excess energy of hot hole is crucial to suppress nonradiative losses associated with surface trapping. As such, high energy excitation in NC lasers and energetic hole injection in LEDs should be avoided to minimize undesired surface trapping, important for wider technological deployment of semiconductor NCs in applications.33. A multipass Ti:sapphire laser  is used to drive both an optical parametric amplifier to create a tunable energy ultrafast optical pump and to excite a photocathode to drive the electron bunch pulses. The electron bunch probe pulses are accelerated to 3.7 MeV to achieve ~200 fs pulse widths with 50 fC charge per pulse. Diffracted electrons were detected using an EMCCD via a red phosphor. Time zero was calibrated for using either thin single-crystal silicon or bismuth samples.UED experiments were conducted at the SLAC National Accelerator Laboratory MeV-UED instrument, a part of the LCLS User Facility. The experimental setup and our analysis approach have been detailed before8 and drop-cast onto TEM grids. Full synthetic and characterization details for the quantum dot stock solutions can be found within ref. 8, with details in Supplementary Section High-quality samples of core-only CdSe and eight ML core-shell CdSe/CdS were used from the same batches detailed in ref. To characterize biexciton Auger lifetime, we measured the core/shell sample under 510 nm excitation. For this, we used a 35 fs amplified Ti:sapphire laser system with a 2 kHz repetition rate. The output of the laser is converted into 510 nm using an optical parametric amplifier. The NC solution  was placed in a 1-mm-thick quart cuvette and excited with varying excitation fluences. The excitation beam size was 496 µm in diameter. The sample was kept stirring throughout the measurement with the help of a small magnet. To capture the photoluminescence decay curves, we used a streak camera (Hamamatsu) providing an instrument response function full-width at half-maximum of 30 ps.50 and a previously implemented interatomic pair potential parameterized for CdSe and CdS51 . Mean square atomic displacements (MSD) for the eight ML core/shell NC were computed from equilibrium MD trajectories at temperatures ranging from 150 to 500 K. A linear relationship between temperature and change in MSD was found and used to estimate experimental transient lattice temperatures.MD simulations were performed on CdSe/CdS core/shell NCs using the LAMMPS code52. Equilibrium MD simulations were used to compute the velocity autocorrelation function for each mode, which was then used to compute the Langevin friction kernel via a numerical Laplace transform and obtain the phonon lifetime (see details in Supplementary Section The phonon density of states was computed for a four ML core/shell NC. The structure was minimized using the conjugate descent algorithm implemented in LAMMPS. This configuration was used to compute the mass-weighted Hessian, which was diagonalized to obtain the phonon frequencies and modes. The lifetimes for each of these phonon modes Fig.  were com53, which uses trajectories with varying time steps to solve classical master equations. The trajectories were initialized with electron–hole pair populations according to the Poisson distribution with the average number of electron–hole pairs consistent with the absorption cross-section and the optical pump fluence. A linear relationship between the amount of electronic energy lost via phonons and the temperature increase of the lattice is assumed. The Auger recombination lifetime was fit to simultaneously reproduce the time dynamics of the time-resolved photoluminescence and ultrafast electron diffraction data in Fig. The kinetic models consist of sets of coupled differential equations (Eqs. S1–S12 in Supplementary Section Supplementary InformationPeer Review File


When analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr–Purcell–Meiboom–Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites.Our aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples.We compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares–Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated.We found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation (< 30%) in replicate samples.Overall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.The online version of this article (10.1007/s11306-020-01686-y) contains supplementary material, which is available to authorized users. Human metabolome profiling can lead to a better understanding of disease mechanisms and to potential biomarkers for improved diagnosis  spectroscopy was the technology that kicked off the field and is one of the two main methods used. The technique has inherent quantitative capabilities   as well as pheochromocytoma and paraganglioma (PPGL) were analyzed using each method and discriminated using supervised multivariate statistics. The results from models separating PA from PPGL were used for the comparison of the methods’ performances in group separation. In addition, we assess the performance of the optimal approach in metabolite quantification using maleic acid as the internal quantification standard. Based on the results presented herein, the novel plasma NMR metabolomics procedure developed is expected to yield valuable sample stratification models, as well as metabolic signatures for understanding the underlying biochemical differences involved in the comparison under investigation.2HPO4⋅2H2O) and Sodium azide (NaN3) were purchased from Merck, D2O 99% and MA from Sigma-Aldrich. Standards used  were obtained from Sigma-Aldrich, except for Glycine which was obtained from Scharlab, Alanine from Fluka and Creatinine from BioChemika. Deionized water was prepared using a Milli-Q Advantage A10 Water Purification System from Merck.Di-Sodium hydrogen phosphate dihydrate  to compare MA to TSP. Subsequently, four pooled plasma aliquots were subjected to ultrafiltration and the remaining four to unfiltered sample preparation for NOESY 1D NMR.A set of 435 QC samples were prepared by pooling ~ 450 mL of plasma obtained from 390 anonymized plasma samples. This large volume of plasma was aliquoted, resulting in 1 mL for each QC sample that was subsequently stored at − 80 °C and used for quantification experiments and intra- and inter-batch repeatability assessment of NMR plasma analyses.2O were added separately to sets of three QCs to make up 15 samples. These samples were analyzed by NMR and routine ion exchange chromatography for amino acid analysis (AAA).For the standard addition experiment, 15 plasma samples were prepared by adding 100 μL from each stock solution to 400 μL of QC plasma. Specifically, 100 μL of stock solutions 1 × , 3 × , 5 × , 10 × and dH2O and centrifugation at 2113×g for 5 min to remove glycerol, 1 mL from each aliquot was transferred to the filter outer tube. Filters were centrifuged at 2113×g for 30 min, resulting in ~ 600 μL of filtrate for each sample. These were stored at − 80 °C until analysis with NMR. Before NMR analysis, both filtered and unfiltered samples were thawed at room temperature. A volume of 350 μL was transferred from each sample to an eppendorf safe-lock tube (1.5 mL) to which the same volume of buffer solution was added. Each safe-lock tube’s contents were mixed using a Retsch vortex mixer and centrifuged at 16,100×g, for 5 min. Finally, 650 μL of each sample were pipetted to a NMR tube.The ultrafiltration procedure was performed according to manufacturer’s instructions with an additional washing step , using a commercially available amino acid mixture for calibration and quantification.https://github.com/NickBliz/High-throughput-Untargeted-Plasma-NMR-Metabolomics_Method) and data can be provided upon request.For spectral processing, all spectra were Fourier transformed, manually phase corrected and the chemical shifts referenced to the glucose doublet at 5.22 ppm, using Bruker Topspin version 3.5. As shown in Fig. Principal Component Analysis (PCA) was used as a method to discover trends and detect outliers, whereas Partial Least Squares Discriminant Analysis (PLS-DA) for separating PA from PPGL samples analyzed with each method. For computing the MVA models, the MixOmics , the significance  of the slope coefficient, the Residual Standard Error (RSE) and the prediction error (RSE/average concentration determined by NMR) were all taken into account. A linear model summary is available for every metabolite spiked.LOD/LOQ Limit of detection (LOD) was estimated by multiplying the standard error of y-intercepts of regression lines in the standard addition experiment by 3.3 and dividing by the slope. Limit of quantification (LOQ) was determined by multiplying the LOD by 3.3.Recovery To evaluate recovery, the percentage of difference in mean of three replicate concentrations for six metabolites at four concentration levels in the test solution between the NMR method and AAA was calculated, along with their respective RSD values.Agreement with Amino Acid Assay Bland–Altman comparison plots (based on R package “Bland–Altman–Leh”(Lehnert ”Lehnert ) were usRepeatability QC samples were analyzed in 45 batches (one QC per batch) using the LED method and the resulting spectra were converted to peaks, which were scaled to each MA peak and normalized using PQN and the median spectrum of all QCs as a reference, to evaluate inter-batch repeatability. For intra-batch repeatability, a set of 19 QC samples were analyzed in one day. Inter- as well as intra-batch RSD values were computed for each peak found in the QC samples.The 18 spectra recorded from all methods had a flat baseline and high resolution Fig. , with haScore plots from PCA models calculated using the AMIX and the SPEAQ data of all methods as shown in Fig. Focusing on SPEAQ results, which led to a lower number of both latent variables and misclassifications (Table 2S), the differences based on the PLS-DA VIP list are summarized in Table 4S. LED resulted in PLS-DA-derived signatures, which had more peaks as well as metabolites in common with UF than CPMG did. We therefore concluded that the LED method is a more appropriate approach for large scale metabolomics and we assessed its quantification capabilities.The initial experiment on 8 replicates samples was used to compare TSP to MA signals in filtered and unfiltered samples. The TSP signal in unfiltered samples had a lower intensity and had a much greater peak width than in filtered samples . On the contrary, the MA peak had an peak width of ~ 1 Hz in filtered and unfiltered samples and in all samples the satellite peaks were visible Fig. S. These 2) were above 0.75 for all models, and significant, with a P value less than 0.05, indicative of a linear relationship.Results of the linearity assessment are summarized in Table 5S. All adjusted coefficients of determination  of 20 μM and differences (− 106–65 μM) are low compared to the concentrations of this analyte. Lysine was quantified both using Chenomx and by directly integrating the peak at 3.01 ppm, but overall agreement was low in both cases, with NMR results being consistently lower than AAA when using Chenomx and higher after direct integration. Alanine presented with an average lack of bias, which ranged from − 116 to 82 μM and, just like glycine, small differences compared to this metabolite’s levels  out of all 107 peaks detected had an inter-batch RSD < 30%, whereas 93 (87%) of all peaks had an intra-batch RSD below 30%. Highly variable peaks were either near the detection limit, high intensity or in close proximity to other peaks.1H-NMR spectroscopy for large studies, with the aim of separating groups of samples on the basis of untargeted metabolome differences. We compare the performance of the proposed approach to the commonly used CPMG method, on the basis of detected NMR signals and sample classification using multivariate statistics, using the UF as a gold standard for the comparison. For large studies, UF is not applicable but when comparing LED to CPMG spectra, an important result found was the increased number of detectable metabolites in LED spectra combined with a more efficient suppression of macromolecular signals. This is the first time that LED is shown, experimentally, not to suffer from these known  and interpretation is more direct, because a fewer number of peaks arise from multiple metabolites. These advantages are of particular importance for building supervised classification models since their accuracy (and thus their usefulness for making predictions) is dependent on the amount of variation explained that is relevant to the biological question Supplementary file2 (TIFF 4433 kb)Supplementary file3 (JPG 3548 kb)Supplementary file4 (TIFF 4433 kb)Supplementary file5 (DOCX 17 kb)Supplementary file6 (DOCX 13 kb)Supplementary file7 (DOCX 13 kb)Supplementary file8 (DOCX 13 kb)Supplementary file9 (DOCX 14 kb)Supplementary file10 (DOCX 13 kb)Below is the link to the electronic supplementary material.


These novel compounds reacted photochemically in the presence of supercoiled plasmid DNA. Structure–activity relationship (SAR) studies revealed that the substituent on the imine group was not affecting the extend of the DNA damage, whereas the substituent of the carbamate group was critical, with the halogenated derivatives to be able to cause extensive single and double stranded DNA cleavages, acting as “synthetic nucleases”, independently of oxygen and pH. Calf thymus–DNA affinity studies showed a good-to-excellent affinity of selected both active and non-active derivatives. Preliminary theoretical studies were performed, in an effort to explain the reasons why some derivatives cause photocleavage and some others not, which were experimentally verified using triplet state activators and quenchers. These theoretical studies seem to allow the prediction of the activity of derivatives able to pass intersystem crossing to their triplet energy state and thus create radicals able to damage DNA. With this study, it is shown that oxime carbamate derivatives have the potential to act as novel effective photobase generating DNA-photocleavers, and are proposed as new leads for “on demand” biotechnological applications in drug discovery and medicine.A number of  Small organic molecules able to bind DNA provide promises for anticancer activity due to alteration of the structure and function of the genetic material. Amongst a plethora of such binders –8, varioThe interaction of molecules with DNA and their affinity towards this macromolecule also plays a key role in photosensitization techniques. Light activated oxidative DNA cleaving agents are called “DNA photocleavers”. These compounds are able to absorb light and to be selectively excited. A variety of reaction mechanisms are initiated, which, aiming to the photocleaver, may lead to DNA damage. A requirement for nucleic acid’s and most protein’s “transparency” is irradiating at wavelengths longer than 310 nm . “Transpa]quinoxaline triazolo radicals, nevertheless, their damage towards DNA was not well recognized and, thus, DNA photocleavage aimed by nitrogen-centered stable organic radicals has been less investigated. Reports involve arylaminyl radicals (ArNH∙) formed from arylhydrazones through the photoinduced cleavage of N–N single bonds  [∙] are formed from the photocleavage of the N–O bond of N,O-diacyl-N-phenylhydroxylamines, along with a carbonyloxyl radical  (Kb constant of 11  = 53.40 kcal/mol, which corresponds to a wavelength of 535.6 nm (visible region). The ground state (S0) bond dissociation energies (BDE), D0, for both molecules under consideration, are shown in These observations differentiate the two molecules giving them different photochemical characteristics and properties. We note that the adiabatic excitation energy of 0 − D0 is higher for 12 than 11 someone would expect 12 to be more reactive in the T1 state as compared to 11, something which is confirmed from our calculations shown below.The BDE difference between the two compounds is about 10 kcal/mol, while that in their adiabatic excitation energies is 3.9 kcal/mol. Since Δ1 FC point, 12 having enough energy reaches T1 min. From there, dissociation of the N–O bond starts to take place and proceeds uphill through a transition state, T1(TS), the structure of which was optimized fully. Vibrational analysis gives a single imaginary frequency (−550.12 cm−1) which corresponds to the N–O bond stretching confirming that the optimized structure is a first order saddle point. Relaxed PES scan was performed until complete dissociation to the respective ground state radicals occurred. In 12 in the T1 state with respect to the reaction coordinate which is the N–O bond distance, rN–O.It is well known that compounds characterized by triplet energies higher than BDEs should exhibit high reactivity –79. Afte12 in the triplet state, the molecule passes through a very small energy barrier and dissociates finally into its photoproducts which are the two ground state radicals indicated in For the dissociation reaction of compound 1 min to T1(TS) is reflected in the low value of the activation entropy, ΔS≠ = 0.474 cal/mol∙K, which means that there is a very small disorganization in the transition state as compared to the reactant in the T1 minimum state. The length of the bond N–O in the transition state is equal to 1.629 Å.The almost negligible change in geometry on going from T12 and found 3.14 and 2.95 kcal/mol, respectively. These values were used in kr, was found to be 4.27 ∙1010 s−1, which is a very large value, indicating a fast N−O bond dissociation for compound 12. Equations 1–3 are described in the theoretical calculations section.The corresponding activation energy  and freep-chlorocarbamoyloxyl) are formed, the second radical starts to decarboxylate according to the chemical reaction below  forces the molecule to pass over an energy barrier of approximately 7 kcal/mol dissociating further and giving ground state radicals again. For the photodissociation of 11 from either electronic excited state  calculations show that this is not possible since in this case the intervening energy barriers are very large, 29.37 kcal/mol for T1 and 31 kcal/mol for the S1 state. Hence, 11 does not show any photoreactivity, in accordance with our experimental results.Excitation of Triplet photosensitizers (TPSs) or triplet state energy activators are compounds that have the ability to be efficiently excited to their triplet excited state. TPSs may be used, among others, to transfer their triplet energy to other molecules that have a low yield of intersystem crossing (ISC) and inefficient production of triplet state ,80. Acet12  we have designed the following experiment: We hypothesized that, upon selective excitation oxime carbamates 8–13 might be excited at their triplet states via triplet state energy transfer from acetophenone as a sensitizer, dissociate to their iminyl/carbamoyloxyl and subsequent anilinyl radicals, attack DNA and cleave it  and carotene (CR), which exhibit low triplet state energy   and may,12 gradually decreases (wells 4–6) until it is totally eliminated (well 7). Additionally, in the presence of CR as a quencher, the energy of the triplet state of 12 is transferred to CR and DNA is not damaged (well 10).It is obvious that in the presence of increasing concentrations of FL the activity of p-Pyridyl oxime carbamates bearing electron-donating or electron-withdrawing substituents on the aromatic carbamate group and an amine, a methyl or a hydrogen on the imine moiety, were successfully synthesized in high yields and subjected to UV irradiation in the presence of plasmid DNA. Not all derivatives were active, nevertheless all amidoxime, ethanone oxime and aldoxime derivatives showed similar effects, indicating the cause of action to be considered on the carbamate moiety rather than the oxime. The affinity of selective compounds with DNA was good to excellent, as verified with extensive CT DNA-affinity studies.11) and the chloro (12) derivatives using UV spectroscopy in the absence of DNA was indicative of the disruption of 11 whereas 12 showed a gradual formation of one product. A theoretical photochemical dissociation study resulted to the observation that 12 has the ability to dissociate, as expected, with homolysis of its N–O bond overcoming very low energy barriers with high rate constants, on the contrary to 11, where its photochemical activity seems to be located at the nitro group. The explanation of the activity seems to be the ability of 12 to obtain its triplet state and create active radicals able to abstract hydrogen atoms from DNA and cause its damage. This was experimentally verified using acetophenone, as a triplet state sensitizer, which transferred its energy to inactive oxime carbamates and enabled them to damage DNA. Additionally, the activity of compound 12 was diminished when triplet state quenchers as fluorenone and carotene were introduced into the irradiated DNA mixture.A deeper insight in the photochemical behaviour of the nitro  DNA, ethidium bromide (EB), NaCl and trisodium citrate were purchased from Sigma–Aldrich Co. CT DNA stock solution was prepared according to standard procedures . The CT –1cm–1 .High-resolution mass spectra (HRMS) were recorded on micrOTOF GC–MS QP 5050 Shimadzu single−quadrupole mass spectrometer. UV–visible (UV–vis) spectra were recorded on a Hitachi U-2001 dual beam spectrophotometer. Fluorescence emission spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer. Viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle and the measurements were performed at 100 rpm. All samples were irradiated with Philips 2 × 9W/01/2P UV-B narrowband lamps at 312 nm.1H and 13C, respectively) spectrometer using CDCl3, and/or DMSO-d6 as solvent. Chemical shifts are given in ppm and J values in Hz using solvent as an internal reference. All reactions were monitored on commercial available pre-coated TLC plates (layer thickness 0.25 mm) of Kieselgel 60 F254. Yields were calculated after recrystallization.Melting points were measured on a Kofler hot-stage apparatus or a melting point meter M5000 KRÜSS, and are uncorrected. FTIR spectra were obtained in a Perkin–Elmer 1310 spectrometer using potassium bromide pellets. NMR spectra were recorded on an Agilent 500/54  were calculated by the Wolfe–Shimer equation [The binding constants of compounds equation  using UV11 and 12  [The viscosity of CT DNA ([DNA] = 0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) was measured in the presence of increasing amounts of compounds 11 and 12 to displace EB from its DNA–EB conjugate was investigated by fluorescence emission spectroscopy. The Stern–Volmer constant  was used to evaluate the quenching efficiency for each compound according to the Stern–Volmer equation [The ability of compounds equation .O-carbamoyl oximes were incubated with supercoiled circular Bluescript KS II DNA and irradiated at 312 or 365 nm for 30 min and 2 h, respectively. The mixture was subjected to electrophoresis, the gel was visualized by 312 nm UV transilluminator and photographed. Finally, quantitation of DNA-cleaving activities was performed using the program “Image J” available at the site http://rsb.info.nih.gov/ij/download.html and the single-strand (ss%) as well as double-strand (ds%) damage was calculated, using a correction factor of 1.43 [All synthesized  of 1.43 .11 and 12 was studied using the density functional theory (DFT) method [The structures, properties and the basic photochemistry of compounds ) method –89 and t) method . Further12 in radicals. The corresponding activation energy and free energy of activation are given in Equations 1–5 were used for the calculations of the rates and the physicochemical data of the N−O bond dissociation of the most active compound kr, the Eyring’s classical kB is the Boltzmann’s constant (1.380662∙10−23 J/K), h is the Planck’s constant (6.626176∙10−34 J∙s), R is the universal gas constant  and T is the absolute temperature in K.For the calculation of the rate constant, Improvement of the Eyring’s equation, incorporating the quantum mechanical tunnelling coefficient, κ, was also used, Q≠ and QR represent the molecular partition functions in the transition state and in T1 min with their values being 0.241907∙1024 and 0.184606∙1024, respectively. Additionally, In ≠) = 5.74672∙1020 J−1 and β = 1/kB∙T = 2.42928∙1020 J−1 are defined through the fundamental constants, ν≠ is the imaginary frequency of the transition state corresponding to that normal mode of vibration which takes the transition state towards the product without a restoring force. In our case, ν≠ = 550.12i cm−1 and Im(ν≠) = 550.12 cm−1 = 1.64921∙1013 s−1, which is a real number.In Finally, the N−O bond energies were calculated according to previously reported methods .1H NMR and 13C NMR of amidoxime, ethanone oxime and aldoxime carbamates; 3) Optimized structures of compounds 25–27 with the ab initio DFT computational methods, 4) DNA binding studies; 5) UV absorption spectra of all amidoxime, ethanone oxime and aldoxime carbamates; 6) Gel electrophoresis pictures of all amidoxime, ethanone oxime and aldoxime carbamate; 7) UV absorption spectra of amidoxime carbamates 11 and 12 under irradiation; 8) A computational study and photochemical aspects of compounds 11 and 12.The Supporting Information features 1) general procedures for the synthesis of all compounds and data analysis; 2) File 1Experimental part.


The tephra layers known with eruption ages play an important role in an investigation of tsunami history and archaeology in addition to volcanic history in Hokkaido, Japan. We investigated the event and tephra layers of the Late Holocene in the Pacific coast of western Hokkaido, where the stratigraphy of the Late Holocene has not been clarified. Surveys in coastal peatlands, mostly undisturbed deposits, have allowed for the discovery of thin tephra layers. The newly discovered tephra layers at the unexplored site were used to describe facies, observation under a polarization microscope, refractive index measurement of volcanic glasses, and chemical analysis, and correlated with the reported widespread tephras. We conducted wide-area field surveys and succeeded in revealing a wider distribution of tephra layers than previously known. The distribution of volcanic ash in the coastal area will contribute to the investigations of future volcanic and coastal hazards. We describe the correlation of each tephra for each region where the combination of tephra layers is similar. In Kameda Peninsula and western Uchiura bay region, volcanic ash layers of Komagatake and B-Tm were identified. B-Tm was easily determined from the unique chemical composition (high potassium) and fine and good sorting grain at all sites. Komagatake tephras (Ko-d and Ko-c2) at Sites 2 and 3, which were difficult to identify from the stratigraphy and mineral compositions, were identified by the scattered plots of SiO22.1A total of 431 samples from cores and outcrops were obtained at the 42 sites along the Pacific coast by using a handy Geoslicer and a Peat Sampler (diameter of 7 cm) with lengths of either 0.6 m or 2.5 m. Photographs were taken of each core sample, and sedimentary facies  were described.2.2To separate volcanic glass and rock-forming minerals from the clay, each sample was vibrated in an ultrasonic cleaning device and the clay components were removed with water elutriation. Samples were dried at 70 °C. The dried samples were divided into 0.063–0.125 phi and 0.125–0.25 phi using a sieve. Refractive index measurements and microscopic observations were conducted for samples of 0.063–0.125 phi and 0.125–0.25 phi, respectively. The samples measuring the glass refractive index were dehydrated by annealing at 400 °C for 12 h in an electric furnace 2.3−4 and a precision of ±1 × 10−4At least 200 grains were counted under a polarization microscope to examine the mineral composition. The refractive index of volcanic glass shards was measured with a Refractive Index Measuring System . This method is possible to measure with an overall accuracy of ±2 × 102.4Chemical analysis of volcanic glass was performed using a JEOL JXA 8900R Electron Probe Micro Analyzer (EPMA) and a JEOL JSM-T330A (Link ISIS300) Energy Dispersive X-ray Spectrometer (EDS). EPMA operating conditions were 15 kV acceleration voltage, 7 nA beam current, beam scanned area of 10 μm, and counting time was 10–60 s at the peak position and 5–30 s at the background-position The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


In citrus, a classical method of studying crossovers and segregation distortion (SD) is the genetic analysis of progenies. A new strategy combining fluorescence-activated cell sorting and whole genome amplification of haploid pollen nuclei with a large set of molecular markers, offers the opportunity to efficiently determine the frequency of crossovers and the identification of SD without the need to generate segregating populations. Here we have analyzed meiotic crossover events in a pollen nuclei population from “Eureka” lemon and the allelic SD was evaluated in a pollen nuclei population from a clementine × sweet orange hybrid (“CSO”). Data obtained from the “CSO” pollen nuclei population were compared to those obtained from genotyping of a segregating population (“RTSO”) arising from a hand-made sexual hybridization between diploid non apomictic selected tangor  as female parent pollinated with “CSO” tangor as male parent. The analysis of crossovers rates on chromosome 1 revealed the presence of up to five crossovers events on one arm and four on the corresponding other arm, with an average of 1.97 crossovers per chromosome while no crossover events were observed in five “Eureka” lemon pollen nuclei. The rate of SD observed in “CSO” pollen nuclei (13.8%) was slightly lower than that recovered in the “RTSO” population (20.7%). In the pollen nuclei population, SD was found on linkage group (LG) 2, while the “RTSO” population showed SD on LGs 2 and 7. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. This methodology is a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD. Most plant species that reproduce sexually alternate the life cycle between a diploid sporophytic phase and a reduced gametophytic haploid phase . MeiosisOn the other hand, allelic segregation distortion (SD), that is, the deviation of the allelic segregation ratio from the expected Mendelian-ratio, is increasingly recognized as an additional powerful evolutionary force . SD may Arabidopsis spp., wheat, barley, maize, tomato, and rice  as basic chromosome number  technique  coupled Citrus limon (L.) Burm. f.] and the allelic SD was evaluated in a pollen grain population derived from a clementine × sweet orange hybrid . Data obtained from pollen grain population were compared to those obtained from genotyping of a segregating population recovered from a hand-made hybridization between diploid non apomictic selected tangor (C. reticulata × C. sinensis) as female parent pollinated with “CSO” tangor as male parent.In the current study, we used a strategy combining flow-sorting of pollen nuclei with WGA and genotyping of individualized nuclei by simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) molecular markers. The meiotic crossover events were investigated in a pollen grain population derived from “Eureka” lemon [Plant material was sampled from the parental collection of our triploid breeding program  carried C. aurantium L.) as female parent and citron (C. medica L.) as male parent  as female parent pollinated with “CSO” tangor as male parent, hereafter referred as “RTSO” population. This hybridization is of great interest in citrus breeding programs to combine desired attributes from both parents such as late maturing from “RTO” and anthocyanin content from “CSO.” Isolated and whole-genome amplified pollen nuclei from “CSO” tangor were genotyped to be compared with the diploid hybrids belonging to the “RTSO” population. Further information about recovery and ploidy level analysis of regenerated diploid plants can be found in On the other hand, for the SD analysis, we used a progeny of 86 diploid hybrids arising from a hand-made cross between diploid non apomictic selected tangor  following the manufacturer’s protocol and measured using a spectrophotometer . The samples were diluted with sterile water  at a concentration of 10 ng/μl and stored at −20°C until use.® filters, Partec®). With the help of a plastic rod, collected pollen grains over the bursting filter were rubbed against the filter and washed with nuclei isolation buffer, twice. Subsequently, DAPI staining solution (1.5 μg/ml) was added to the suspension and incubated for 10 min. Stained suspension were run in a BD Influx  and analyzed with BD FACS software.For pollen grain recovery, between 40 and 50 flowers of the “CSO” tangor and “Eureka” lemon were collected in pre-anthesis during the spring of 2018. The anther’s were removed from the flowers with forceps and were dried in Petri dishes over silica gel in a desiccator at room temperature until the anthers opened after 1 or 2 days. Then, dehiscent anthers were selected under magnifying glass, discarding those that were not fully open, and they were sealed with parafilm and stored at −20°C until use. To isolate pollen nuclei we used the methodology described by 2O, and 1 μl sample buffer . Illustra GenomiPhi V2 DNA Amplification Kit  was used to WGA Prior to calculate the DNA concentration of WGA products, each sample was diluted with 500 μl of ddH2O, and subsequently was measured by fluorometric quantification .We have followed the methodology described by Fifty-four pollen nuclei of “CSO” tangor and 44 pollen nuclei of “Eureka” lemon were sorted and whole genome amplified. Additionally, 10 single pollen nuclei of the analyzed genotypes were mixed in the same well to be used as a positive control against amplification errors .The hybrids of “RTSO” progeny and their parents together with the amplified pollen nuclei of “CSO” tangor were genotyped using 30 molecular markers (23 SSRs and seven SNPs) heterozygous for “CSO” and with polymorphism with “RTO.” These markers are distributed across all linkage groups (LGs) of the Clementine genetic map . Isolate®) using the following protocol: reaction volume, 15 μl containing 0.5 μl of 1 U/μl of Taq DNA polymerase (Fermentas®), 3 μL of citrus template DNA (10 ng/μl), 1.5 μl of 2 mM welled (Sigma®) dye-labeled forward primer, 1.5 μl of 2 mM non-dye-labeled reverse primer, 0.2 mM of each dNTP, 1.5 μl of PCR reaction buffer 10×, and 0.45 μl of 50 mM MgCl2. The cycling program was set as follows: denaturation for 5 min at 94°C followed by 40 cycles of 30 s at 94°C, 30 s at 50°C or 55°C, 30 s at 72°C; and a final elongation step of 8 min at 72°C. Separation was carried out by capillary gel electrophoresis using a Genetic Analysis System 8000 (Beckman Coulter Inc.). The PCR products were initially denatured at 90°C for 2 min, injected at 2 kV for 30 s, and separated at 6 kV for 35 min. Alleles were sized based on a DNA size standard (400 bp). GenomeLabTM v.10.0 (Beckman Coulter Inc.) genetic analysis software was used for data collection.Polymerase chain reactions (PCRs) using SSR markers were performed using a Thermocycler rep gradient S -based assay for SNP genotyping, and detailed explanation of specific conditions and reagents can be found in For SNP markers genotyping we used KASPar2), assuming an expected allelic segregation ratio of 1:1 (p < 0.05) for each analyzed marker.The potential allelic SD was analyzed in the “RTSO” progeny as well as in the pollen nuclei population from “CSO” tangor using the Chi-square test  is the number of crossovers and n (M) is the total number of observations. The pairs of markers in the analyzed gametes that showed missing data points were omitted from the analysis.where Population diversity organization between hybrids of the “RTSO” progeny at the male gamete level and the “CSO” pollen nuclei population was examined using DARwin6 software . Neighbodi–j is the dissimilarity between units i and j, L is the number of loci, ml is the number of matching alleles for locus l, and π is the ploidy level, which is one in this case. From the dissimilarity matrix obtained, a Weighted neighbor-joining tree  to establish the reference citrus haploid whole genome sequence  with no crossover events between analyzed markers , resultisequence .Hordeum vulgare) (Arabidopsis (Zea mays), both low temperature and water deficit significantly enhance the frequency of crossovers  , whereasbidopsis . Similarossovers .C. clementina where centromeric areas showed low recombination rates (<1.0 cM/Mb), although this reduction changed greatly between chromosomes  was slightly lower than that recovered in “RTSO” population (20%). In the pollen nuclei population, SD was found on LGs 2 and 5, while the “RTSO” population showed SD on LGs 2 and 7. Globally, a total of seven analyzed markers showed SD. Among these markers, 2P21022555, CX6F23, and CIC3712-01, all located on LG 2, were synchronously distorted in the two populations. However, MEST88 marker, located on LG 5, shows SD for the pollen nuclei population, and not in the “RTSO” progeny. In addition, MEST107, MEST202, and CIC3674-02 markers, all located on LG 7, showed SD only in the “RTSO” population.Mimulus sp.), cotton (Gossypium hirsutum) it has been observed gametal factors that influence male gametes viability  as we have observed in both populations of “CSO” male gametes on LG 2. One explanation for SDRs could be that specific loci in the genome are conducted to viability differentiation . The selC. sinensis as the same ancestor and could share a S-RNase haplotype, whereby segregate as could be expected for the GSI system. These results could also suggest that in this genomic region could be located alleles related with the GIS in citrus. In fact, Likewise, a strong SD has been observed on LG 7 only in “RTSO” progeny rather than “CSO” pollen nuclei population . The gamOn Drosophila obscura  or with male and female gametic interactions, or zygotic mechanisms (LG 7). In addition, this methodology may have an important advantage for the achievement of sequencing projects. High heterozygosity is a general characteristic of Citrus species, making it difficult to assemble large genome sequences. For this reason, the ICGC decided to establish a reference whole citrus genome sequence from a clementine homozygous plant recovered by Fluorescence activated cell sorting coupled with WGA allows the analysis of individual nuclei with a large number of molecular markers, offering the opportunity to efficiently determine the frequency of crossovers and the SD in haploid pollen nuclei without the need to generate segregating populations.Until now, no study was conducted regarding the recombination in citrus pollen nuclei. The analysis of the “Eureka” lemon pollen nuclei allowed the identification of recombination points through the use of SSR and SNP markers, showing a greater number of crossovers in centromere distal regions of chromosome 1. In addition, SD has been observed either on LG 2 of both populations and only on LG 7 we have observed that the SD in the pollen of “CSO” tangor and the plants of the population differ in frequency or position. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. The methodology presented here represents a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD.All datasets generated for this study are included in the article/MG and PA conceived the study and were in charge of the direction and planning. JF isolated pollen nuclei and conducted flow-sorting. SD performed whole genome amplification from single pollen nuclei. MG, JC, and PA genotyped pollen nuclei, diploid hybrids, analyzed the data, and took the lead writing the manuscript with input and review of LN, SD, JF, and AH. All authors read and approved the final version of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Programming deep brain stimulation in dystonia is difficult because of the delayed benefits and absence of evidence-based guidelines. Therefore, we evaluated the efficacy of a programming algorithm applied in a double-blind, sham-controlled multicenter study of pallidal deep brain stimulation in dystonia.A standardized monopolar review to identify the contact with the best acute antidystonic effect was applied in 40 patients, who were then programmed 0.5 V below the adverse effect threshold and maintained on these settings for at least 3 months, if tolerated. If no acute effects were observed, contact selection was based on adverse effects or anatomical criteria. Three-year follow-up data was available for 31 patients, and five-year data for 32 patients. The efficacy of the algorithm was based on changes in motor scores, adverse events, and the need for reprogramming.n = 17) during the monopolar review. Contacts without acute benefit improved by 58 ± 30% at 3 years (n = 63) and 53 ± 31% at 5 years (n = 59). Interestingly, acute worsening or induction of dystonia/dyskinesia (n = 9) correlated significantly with improvement after 3 years, but not 5 years.The mean (±standard deviation) dystonia motor score decreased by 73 ± 24% at 3 years and 63 ± 38% at 5 years for contacts that exhibited acute improvement of dystonia (Monopolar review helped to detect the best therapeutic contact in approximately 30% of patients exhibiting acute modulation of dystonic symptoms. Acute improvement, as well as worsening of dystonia, predicted a good long-term outcome, while induction of phosphenes did not correlate with outcome.NCT00142259.ClinicalTrials.gov  Primary dystonia comprises a heterogeneous group of incurable, idiopathic movement disorders with involuntary muscle contractions leading to twisting, repetitive movements and abnormal postures , 6. OralThe reported benefits from GPi-DBS include a 50–80% reduction in dystonia motor symptoms , 11, 13.The original trial was a double-blind, sham-controlled study for 3 months, followed by an open-label extension for up to 5 years, including 40 patients with pharmacologically-intractable, primary generalized or segmental dystonia . At implThe algorithm to determine the active stimulating electrode was defined in the study protocol. Within the first week of implantation, a monopolar review of all electrodes of the quadripolar lead (Medtronic 3387 or 3389) was performed. With the IPG as anode (+) and stimulation frequency and pulse width kept constant at 130 Hz and 120 μs, respectively, each electrode, beginning with the most distal one, was stimulated as monopolar cathode (−) with an increasing amplitude to a maximum of 6 V for 60–90 s, as long as no acute adverse effects were elicited. Induction of beneficial effects  or adverse effects  were documented. If beneficial effects were noted, the respective electrode was chosen for long-term stimulation. Otherwise, an electrode that elicited phosphenes at an amplitude above 3 V was selected. If phosphenes were induced below 3 V, the next proximal electrode was chosen. At the time of planning the study (around 2000), imaging and image fusion techniques to reconstruct the individual electrode positions postoperatively were not readily available, neither were volume of tissue activated (VTA) models established. Acute clinical response testing was used to establish the anatomical position of the electrodes and based on expert consensus, we considered a phosphene threshold below 3 V as indicating a proximity to the optic tract, indicating a contact location below the ventral border of the GPi. Testing was suspended above 6 V, because higher settings were deemed to be inappropriate for chronic stimulation without causing adverse effects.If neither beneficial effects nor optic tract response (= phosphenes) could be elicited through any electrode of the lead, the most distal electrode presumed to be located in the ventral GPi, based on intraoperative microelectrode recordings (MER) and/or pre- and postoperative magnetic resonance imaging (MRI), was activated (anatomic choice). Corresponding to the monopolar review, stimulation frequency and pulse width were initially set to 130 Hz and 120 μs. In the neurostimulation group, amplitude was set 0.5 V below the threshold of eliciting adverse effects from the start of the monopolar review, or up to 6 V if no side effects were elicited . Where appropriate, results are presented as mean ± standard deviation.A total of 27.8% of active contacts were chosen because of acute improvement in dystonic symptoms during the monopolar review. Another 30.4% were chosen because of elicitation of phosphenes. The majority of contacts (41.8%) were selected anatomically (based on nuclear boundaries determined by intraoperative MER and/or MRI), since neither acute effects nor phosphenes could be elicited.During the initial 6 to 9 months study period, 85% of the electrode configurations remained compliant with the PA; 67% were still compliant at the three- and five-year follow-up. We considered the configuration still compliant if another electrode was added (double monopolar configuration) adjacent to the original one.The method of choice for the primary contact selection did not have a significant impact on maintenance of the stimulated contacts, i.e. whether based on anatomical aspects (50%), optic-tract response (23%), or acute stimulation benefit (27%). However, there is evidence for a trend towards higher compliance with contacts chosen on an anatomical basis.n = 44 contacts) vs. 53 ± 34% if there was a deviation (n = 36 contacts). This difference was not significant.At the five-year follow-up, the mean improvement in the BFMDRS score was 57.6 ± 32% if the electrode selection was compliant with the algorithm (n = 17 contacts) and 63 ± 38% after 5 years (n = 21 contacts). This compared to 58 ± 30% after 3 years (n = 63 contacts) and 53 ± 31% after 5 years (n = 59 contacts) in patients without an acute antidystonic effect. The difference was significant after 3 years (p < 0.05), but not after 5 years.Electrodes exhibiting acute improvement of dystonia during the monopolar review led to an average decrease in the motor score of 73 ± 24% after 3 years  during the monopolar review were associated with significantly better reduction in the motor score (78 ± 11% after 3 years) compared to 59 ± 31% for all other electrodes . Electrodes selected based on this kind of acute modulation of dystonia showed significant better response on the BFMDRS after 3 years  compared to those selected anatomically  or based on the optic-tract response  , the outcome was still more favorable with a BFMDRS improvement of 61 ± 32% for electrodes with acute modulation of dystonia vs. 54 ± 33% in the remaining, but no longer significant.Interestingly, electrodes that evoked acute dyskinesia or worsening of dystonia without inducing capsular side effects  and a favorable, but non-significant outcome at 5 years (68 ± 22% vs. 53 ± 34%). Other acute adverse effects reported during the monopolar review, such as dysesthesia, visual sensations, nausea, tetanic muscle contractions, or subjective changes in tension and pain in the dystonic body region, had no bearing on the long-term motor benefits.Dysarthria was another acute stimulation-induced adverse effect during the monopolar review that was associated with a significant better outcome at 3 years  after 3 years compared to contacts eliciting phosphenes  or selected on anatomical grounds   showed significantly better improvement of motor outcome with a mean decrease in motor score of 77 ± 16%  of electrodes after 6 months and in 15.8% at 3 years and 5 years (both n = 25) for two reasons: (1) trying to improve efficacy in poor or non-responders ; (2) trying to control stimulation-induced adverse effects . The most common specific reasons for the stimulation changes documented in the case report forms were “worsening of dystonic symptoms” (n = 9) and dysarthria (n = 7).A change in the active contact was clinically initiated in 19.6%  may be well invested in dystonia, even if a smaller proportion of patients can be programmed based on immediate beneficial feedback compared to Parkinson’s disease.Another third of the electrodes was selected based on stimulation-induced visual phenomena (perception of light/phosphenes) according to our algorithm. This criterion was chosen because this stimulation effect indicates proximity of the electrode to the optic tract, which runs a few millimeters below the target region of the GPi. We found no difference in clinical outcome between the electrodes chosen by visual stimulation effects and those selected on anatomical grounds. Hence, eliciting visual phosphenes provides rough confirmation of anatomically correct positioning of the electrode, but cannot be regarded as a predictive marker for an excellent clinical response , 8.Interestingly, we found that not only acute improvements in dystonia, but also stimulation-induced worsening of dystonia or induction of dyskinesia were associated with an above average outcome. Therefore, any modulation of dystonia during a monopolar review should be regarded as predictive of long-term efficacy; patients can be encouraged to tolerate transient worsening of their condition with the perspective of an excellent outcome.Among the other acute adverse effects elicited during the monopolar review, only dysarthria predicted a better outcome at 5 years. This is remarkable, because dysarthria is considered an intolerable adverse effect of pallidal stimulation and programming strives to minimize the risk. However, our finding indicates that the cortico-bulbar fibers causing dysarthria may run within the internal capsule in close proximity to the antidystonic “sweet spot” within the GPi and can therefore guide the selection of an optimal electrode, if a sufficient adverse effect threshold is respected.In summary, the observations made in this study have the potential to change the clinical approach to programming DBS for dystonia. In a monopolar review, clinicians should search for any acute change in dystonia, either improvement or induction of hyperkinesia, which is predictive for an excellent long-term outcome. These acute modulations of dystonic symptoms were significant better outcome predictor than phosphene, which so far were interpreted as a good outcome predictor, due to the close proximity of the optic tract and ventral internal pallidum. Paradoxically, a low threshold for dysarthria may also indicate an electrode that should be stimulated for optimal outcome below the adverse effect threshold. If none of these acute effects is observed, then programming should be guided anatomically. Recent advances in image processing and software technology provide options for fast and accurate electrode location within the individual anatomical space, which may facilitate this anatomical selection in the future.