However, δ2Hn assays of organics are usually complicated by uncontrolled “exchangeable hydrogen” and residual moisture contamination; hence, δ2Hn assays are persistently incomparable amongst laboratories. We introduce a revised technical solution (UniPrep2) to control hydrogen-isotope exchange and for robust online sample drying and vapour equilibration. The UniPrep2 device is coupled to a high-temperature thermochemical elemental analyser and continuous-flow isotope-ratio mass spectrometer. This technical solution empowers isotope analysts to:•δ2Hn values by controlling hydrogen-isotope exchange.Conduct reproducible controlled vapour equilibrations of complex organic samples and standards to determine the •Conduct online vacuum-oven evacuation with extensive helium drying without exposure to air to reabsorb or exchange hydrogen with ambient water vapour.Non-exchangeable hydrogen-isotope  approach, intended for determining δ2Hn for a complex organic environmental sample where matrix equivalent H isotope reference materials are not available, and the Comparative Equilibration (CE) approach, which is intended for routine high-throughput analyses of complex organic samples where at least two matrix-equivalent organic isotope reference materials with consensus δ2Hn values are being used. These standard operating procedures are envisioned to be a sound basis for advancing hydrogen-isotope analysis for different organic environmental matrices and studies.The protocol describes the operation of the UniPrep2 device and the step-by-step procedures needed to obtain accurate and precise   Specifications tableSee Step 10δ2H) measurements of complex organic samples  are increasingly and broadly used in environmental forensics for the determination of sample authenticity and traceability in archaeology, in geology, and in ecological studies of animal migration and resource use δ2Hn) in organic matter is derived from local environmental water and correlates with geographical provenance via predictable hydroclimatic hydrogen-isotope patterns in continental precipitation [2O vapour in laboratory air during sample handling 18O) contamination. Traditional offline sample drying methods to remove residual moisture  are time-sensitive operational techniques  that offer no subsequent quantitative means to prove  that all residual moisture has been removed. Laboratory air can be humid . This is especially concerning when offline dried samples are re-exposed to air for some time during their handling and weighing into capsules. Accordingly, measurements of the δ2H isotope composition of complex organic environmental samples have remained persistently incomparable amongst worldwide laboratories owing to i) inconsistent approaches in controlling and eliminating hydrogen-isotope exchange, ii) variable or poorly reproducible approaches to sample drying that do not quantify elimination of trace residual moisture contamination, and iii) furthermore the unfortunate lack of a wide range of types of complex organic reference materials having well-known δ2Hn values. These three factors combined have seriously undermined the comparability, precision, and accuracy of organic δ2H data produced amongst stable isotope laboratories worldwide, as evidenced by poor outcomes in organic hydrogen-isotope proficiency tests [δ2Hn results amongst laboratories is crucial to the success of hydrogen-isotope provenance, authenticity, and environmental research studies and the development and interoperability of global environmental hydrogen-isotope databases Hydrogen stable isotope  δ2Hn values and control hydrogen-isotope exchange, followed by online vacuum-oven drying with extensive helium flushing and drying δ13C and δ15N determinations of organic and inorganic samples. In short, the UniPrep device enables a vapour equilibration and online vacuum drying schema to control hydrogen-isotope exchange tightly and eliminate residual moisture contamination.In 2015, we introduced a technical solution to address the first two points, uncontrolled hydrogen-isotope exchange and online sample drying, by offering the Universal Preparation device  and Comparative Equilibration (CE)  available and hence require considerable extra analytical effort. By contrast, the Comparative Equilibration (CE) approach is far more straightforward and intended for routine and high-throughput analyses of complex organic samples  where at least two isotopically separated matrix-equivalent H isotope reference materials with consensus δ2Hn values are readily available.The two general approaches used by the UniPrep2 to control hydrogen-isotope exchange in organic samples are known as ion (CE) . The Duaδ2Hn analyses for various classes of complex organic samples for which matrix-equivalent δ2Hn isotopic reference materials with consensus values have not been developed or are unavailable. Recently, a list of hydrogen-isotope reference materials with well-established consensus δ2H values has been made available exƒ) and H mass fraction and (ii) to subsequently derive its δ2Hn. The DVE approach requires two separate analyses of the same sample, each controllably exposed to a sufficiently isotopically distinctive water vapour, after which samples are extensively dried and analysed, preferably online. The extent of δ2H isotopic response of each sample exposed to different vapours is solely a function of the fraction of its exchangeable hydrogen , which can be used to deduce the non-exchangeable hydrogen fraction that is of interest. For example, an organic sample with no exchangeable hydrogen  should exhibit no δ2H response to different isotopic vapour exposures after drying δ2Hn reference materials with well-known consensus values have not been developed. The DVE approach requires concurrent analyses of water references having known δ2H values to determine the δ2Hn of the samples and ensure the results are anchored to the VSMOW-SLAP primary reference scale (see Step 10 below).The most encountered scenario is the need for δ2Hn values on the VSMOW-SLAP scale are used for data normalization and which are reliably quantified for their exƒ values. Under the CE approach, unknown organic samples, their exchangeable hydrogen properties, and hygroscopicity are presumed to have been pre-established by DVE and to be (sufficiently) identical to the consensus hydrogen-isotope reference materials . In this case, a single vapour equilibration and online drying are equally applied to all samples and reference materials in a single UniPrep2 analysis batch. The CE approach has proven to be highly reproducible and robust amongst laboratories, is fast and less laborious than other methods, and hence is a preferable approach, provided that appropriate matrix equivalent reference materials exist [δ2Hn reference materials currently exist having certified values [δ2Hn reference materials are wood powders  and keratin powders  available from https://www.usgs.gov/labs/reston-stable-isotope-laboratory/reference-materials-and-calibration-services. Other new complex organic reference materials intended for eventual CE use for the determination of δ2Hn measurements are under development, and hopefully for a broader suite of organic sample matrices. A decision flowchart for deciding whether the DVE vs CE approach should be used with the UniPrep2 device is shown in The second, more widely-used scenario is the CE approach ls exist ,21,22,26d values ,21,22,26δ2H are routinely published. These approaches include ignoring the hydrogen-isotope exchange issue, reporting organic sample δ2H values against inappropriate  isotope reference materials δ2Hn reference materials not matrix equivalent, thereby guaranteeing that results will be incomparable with other laboratories, as seen for example in It should be acknowledged that unacceptable approaches for organic δ2Hn values in complex organic samples. The UniPrep2 device is compatible with CF-IRMS systems from ThermoFisher , Eurovector , Hekatek (HTC), and with various interface systems  that connect EAs to the UniPrep2 device using a serial connection and automated carousel software trigger. Here we document analytical procedures for the latest UniPrep2 using a ThermoFisher Flash HT EA system connected to a ThermoFisher DeltaV CF-IRMS. It should be noted that our examples are transferable to other HTC EA and CF-IRMS systems with minor technical or software modifications.Since the original UniPrep was launched in 2015 and was incorporated by stable isotope laboratories, feedback from analysts recommended some technical improvements to the UniPrep device as well documentation of optimal and reproducible DVE and CE procedures for the device in connection with HTC EA and the CF-IRMS systems for the determination of •Elimination of some identified cold spots (where vapour could condense) at the bottom of the carousel by removing the motor housing from the base and fully incorporating the EA isolation valve into the heated zone.•A helium-pressurized triple-O-ring motor axel housing to eliminate potential vapour or helium losses from the device during its use .•Addition of a helium purge valve with a capillary outlet that allows users to vent or flush the carousel for sample drying (or as zero-blank) with controlled carousel helium flushing rates from 85 mL/ min (@ 0.8 bar helium pressure) to 150 mL/ min (@ 2 bar helium pressure).•δ2H and δ18O measurements and all crushed tin capsules for δ13C and δ15N measurements.The carousel tray size was increased to 80 (79 usable) samples (from 50 (49 usable)) and further optimized for 3.5 mm O.D. by 3–9 mm silver capsules for  •An improved vacuum system rapidly evacuates the carousel via capillary to <5 mbar pressure (for vacuum vapour equilibration) and for evacuating air and residual moisture.•A viewing glass reveals the internal user-adjustable index counter and a direct view into the EA-HTC reactor to check for sample blockage.•Compact cartridge heaters, a thermocouple, and a reduction in cabling (not depicted).•A helium control valve allows matching carousel helium pressure to the EA system to minimize pressure pulses and carrier flow variations to the CF-IRMS when opening the isolation valve.•Incorporating a vacuum pump, temperature controls, and a helium pressure regulator into a single control box.•Addition of EA connection fittings for 17- or 18-mm O.D. HTC ceramic or quartz reactor tube systems.•Precise isothermal heating and temperature control of the carousel for drying and vapour equilibrations ranging from 40 to 90 ± 0.1 °C or may be used at ambient temperature.Compared to the original UniPrep device, the new 2022 version  incorporδ2Hn measurements of organics are discussed in later sections):•The “Isolation valve” opens/closes the UniPrep2 to the analytical circuit of the EA and the CF-IRMS. When closed, helium carrier gas from the EA flows normally through the EA into the CF-IRMS .•Samples and references are loaded in 3.5 mm O.D. x 3–9-mm silver capsules are placed into the 79-position sample tray .•An adjustable indexing disc  shows th•The “Vacuum Out” valve on the manifold opens/closes the vacuum pump line to the autosampler. When the cover is closed , the vacuum pump evacuates air and moisture from the carousel and samples to < 5 mbar pressure.•A Pressure gauge on the manifold monitors the chamber pressure  or vacuum status .•2O injection septum port” allows users to inject liquid water (up to 150 µL of H2O) into the evacuated chamber for isothermal timed static sample-vapour isotopic equilibrations.The “H•The “Helium In” valve  allows helium to fill or flush the sample chamber and is pressure adjustable to match the Elemental analyser pressure before opening the “Isolation valve” to the CF-IRMS.•The “He purge” valve allows for a constant flush of helium through the sample carousel up to approximately 150 mL/min with the “Isolation valve” closed or allows for venting to open the carousel.•The “Temperature Controller” sets the precise carousel temperature (40–90 ± 0.1 °C) for vapour isotopic equilibrations, outgassing, and online vacuum drying. Optionally, the device can be operated at ambient temperature.Regarding Step 1. Ready the CF-IRMSδ2H stable isotopic analyses per manufacturer specifications, including ensuring the best IRMS focus tuning for low and reproducible H3+ contribution, sensitivity, and stability testing in a continuous-flow mode. Depending on the dynamic range of the IRMS system, the organic samples and reference sample masses should be targeted to achieve roughly the same H2 signal, typically between 6000 and 8000 mV (instrument specific). An ion source linearity test should be conducted by measuring an organic standard by ranging its mass, typically between 200 and 1000 µg, to determine the response of the CF-IRMS to varying H2 yields from sample pyrolysis. In most cases, an ion source linearity correction can be derived using offline data processing software like LIMS for Light Isotopes2 yield-dependant isotopic variance.The CF-IRMS system should be readied for Step 2. Prepare the HTC-EA reactor tubeδ2Hn and δ18O measurements for organics are required. Most EA systems have reactor tube connection fittings for either 17- or 18-mm O.D. ceramic or quartz tubes with lengths ranging between 450 and 470 mm. The correct UniPrep2 EA adaptor should be installed for the selected reactor tube.The HTC pyrolysis reactor configuration will partly depend on the make of EA being used, the reactor tube length, EA fittings, EA reactor temperature range available, and whether δ2Hn measurements of complex organic samples that contain nitrogen  or halogens because the chromium metal catalyzes the removal of CO and nitrogen and reduces bias by potential hydrogen-isotope fractionation due to formation of non-target by-products  2 peaks without any CO or CHx peaks and with minimal N2 peaks for nitrogenous samples.2 chromatography due to lower gas dispersion in the fine chromium powder, 40 % less chromium reagent is used compared to a straight quartz tube, and quartz is transparent for convenient viewing during reagent filling. Moreover, HTC EA oven life is extended by avoiding operation at highly elevated temperatures.The 18-mm-O.D. Quartz capillary chromium reactor , right iδ2H and δ18O) analyses are impossible due to carbon removal by the elemental chromium reagent and lower temperature. Our testing showed that chromium reactor temperatures below 1150 ºC are not recommended, as revealed by CHx and CO peaks increasingly rising at lower temperatures on the Thermal Conductivity Detector (TCD), primarily when chromium reactors are run much lower than 1100 ºC. The EA GC oven should be hot (100 °C) and fitted with a 1.5-m packed “oxygen separation” column to ensure a distinct H2 gas peak. Helium flow rates through the reactor should not exceed approximately 80–90 mL/min to allow complete sample conversion to H2 gas. The EA-TCD should be calibrated and monitored to quantify sample hydrogen yields. Sample run time will be approx. 300–400 s and is EA-specific.One disadvantage of quartz tubes is that dual-isotope  to pre-condition a new C reactor will supply sufficient available carbon to form H2 and CO for later samples. The EA GC oven should be hot (100 °C) with a 1.5 m oxygen separation column to ensure clean separation of the eluting H2, N2, and CO gas peaks. Helium flow rates through the carbon reactor should not exceed 80–90 mL/min to ensure complete conversion to H2, N2, and CO gas δ2H analyses. Some EA configurations offer a reverse helium flow configuration and bottom feed adaptor, which is not needed for δ2H (and reduces sample capacity). A 12.7-mm-O.D. x 2.4-cm thin-walled graphite crucible (Alpha Resources P/N: AR053HD) placed on top of the glassy carbon chips will catch the silver capsules for easy cleanup after 8–10 gs of molten silver has accumulated. After cooling and removing the reactor, the silver “nugget” rolls out of the crucible. The ceramic reactor can be reused for at least 2000–3000 samples before replacing the inert glassy carbon chips. Sample run time will be 300–500 s and is EA and CF-IRMS specific. After each new reactor installation, an automated or manual EA system helium leak check is strongly recommended.The 17-mm-O.D. ceramic reactor , left isStep 3. Preparing the Organic Samples and Referencesδ2Hn, values, the organic samples and references are weighed into silver capsules. They are isothermally and statically equilibrated to either one (CE Method) or two (DVE Method) isotopically known water vapours with sufficient time for full hydrogen-isotope exchange without any hindrances caused by crushing or tightly closing the sample capsules. Silver capsules should be selected for the smallest size adequate to hold the samples and references without overfilling and allow total vapour exposure. Round-bottom “liquid” silver capsules 3.5 mm O.D. x 4–9-mm tall that weigh 70–120 mg each are ideal, compared to thin-walled 14-mg thin-walled EA silver capsules. Organic samples and references are typically weighed out between 200 and 700 μg  into the silver capsules. The masses of the samples and standards should be carefully and accurately H2-yield-matched  to obtain approximately the same EA and CF-IRMS H2 peak height and area to avoid significant ion source linearity deviations. Pre-testing of unknown samples to estimate the hydrogen content may be required. In many cases, biological organic samples or foods may contain significant fractions of 2H-depleted lipids or volatile organic compounds (VOC), which should be removed by using a preparative solvent  removal step, because lipids will especially masque the non-exchangeable hydrogen signal For the determination of δ2Hn measurements and vapour equilibration of complex organic samples was how to ensure and control the sample vapour exposure and later facilitate the complete removal of equilibration vapour and residual moisture for both samples and reference materials. Traditionally, EA samples are tightly encapsulated or crushed into thin-walled silver capsules; hence, hydrogen-isotope exchange and residual moisture removal are exceedingly challenging to guarantee and may even be prevented by blockage and sealing. Previous adaptations that enable vapour exposure for equilibration and drying included loose encapsulation or micro-puncturing of the silver capsules to facilitate vapour and gas exchange 2O exposure, whether tightly or loosely encapsulated, stored on a shelf, or placed in a desiccator.One longstanding challenge for To overcome uncontrolled vapour blockage during equilibration, the UniPrep2 carousel was redesigned to use the robust 3.5-mm-O.D. by 4–9-mm tall round-bottomed ("liquid") high-purity silver capsules with the capsules left fully open . Allowing the silver capsules to remain open-topped inside the carousel ensures that all samples and references are equally and fully exposed to the vapour for hydrogen-isotope equilibration, vacuum drying, and helium drying without any hindrance to isotope exchange or vapour movement. These solid silver capsules cannot tip over and lodge sideways in the carousel and so have a minimal risk of spilling their contents inside the UniPrep2. Moreover, a gentle carousel rotation drops capsules into the HTC reactor without tipping. Evacuation and helium flushing are made through small capillary tubes to avoid abrupt carousel pressure changes .δ2H values are required because the δ2Hn values must be anchored to the VSMOW-SLAP water scale. However, using water for EA standards is almost impossible to dispense and cold-weld into small silver capsules without compromise, and so to be later unaffected by heat or vacuum on the UniPrep2 device. Instead, we strongly recommend using USGS reference waters supplied pre-sealed in silver microtubes. These silver microtubes are unaffected by heating or vacuum in the UniPrep2 carousel and yield highly reproducible results. Notably, we suggest using VSMOW and SLAP or the appropriate δ-value range of pre-calibrated waters provided in 0.15 mL or 0.25 mL silver microtubes by the U.S. Geological Survey . The δ range of reference waters selected for DVE should fully bracket anticipated δ2Hn range of the organic samples after they are exposed to the equilibration H2O vapour (see Equilibration), especially if using highly 2H-enriched or depleted equilibration vapours. For a control standard, one can use water reference materials in silver microtubes or other isotopically proven organic materials – but importantly, the control should not have exchangeable hydrogen. Recommendations for reasonable non-exchangeable controls include NBS22a oil (available in 0.15 or 0.25 mL in silver tubes) or USGS 77, a polyethylene powder that can be weighed into the open silver capsules like the unknown samples. USGS77 is especially useful because it should not respond to any vapour equilibration; hence, isotopic deviations of this control standard from the expected value would show that the carousel may not have been sufficiently dried or helium flushed. The only reference materials required for the CE approach are two matrix equivalent complex organics and USGS77 as a control to correct for instrumental drift and linearity.For the DVE approach, at least two water references with known consensus δ2Hn measurement are pulverized (to maximize equilibration vapour exposure and ensure isotopic homogeneity), but this is entirely dependant on the scientific purpose and the types of sample materials , a typical H2 peak area on the CF-IRMS is 100 vS (8000 mV peak height); hence, the unknown organic samples should have a target weight that produces approximately the same H2 peak area and size. For keratins and wood samples (6–7 % hydrogen by mass), the comparative sample weight needed is around 400 µg, and for USGS77 polyethylene powder (14.5 % hydrogen by mass) is around 200 ug. The recorded sample in the silver capsule is carefully transferred from the microbalance into a 96-well position-numbered plastic plate. This plate with samples and references should be secured with a protective plastic cover for storage until hydrogen-isotope analyses. When storing “open-top” silver sample capsules for later analysis, care must be taken to avoid shaking or tilting the plastic storage plate to avoid capsule tipping and sample spillage inside the tray. Moreover, silver capsules quickly oxidize under moist light conditions; hence, they are best stored under dry and dark conditions or analysed immediately after sample preparation.For weighing an organic sample, an internally round-bottomed silver capsule  is tared using a high-precision  analytical microbalance. The samples are weighed using a clean stainless-steel spatula or cut with a blade to within ± 50 µg of the target weight. The target weight of the organic sample must be pre-determined using its estimated or pre-measured total hydrogen mass concentration, the CF-IRMS peak area of the sample, and the reference materials being used. For example, when 0.25 mL (250 µg) of USGS reference waters in silver microtubes are used . After the first vapour-equilibration sequence, the run is stopped, the UniPrep2 “Isolation valve” is closed, the carousel is evacuated, and followed by injection of the high δ2H vapour for equilibration. The advantage of using the split template is that the DVE process is completed in a single batch with no changes to the CF-IRMS tuning settings or to the chromium or carbon reactor. An alternative approach is to analyse a full carousel with a low δ2H vapour equilibration, clean out the reactor as required, and then repeat the entire carousel with a high δ2H vapour equilibration. Either approach is acceptable.For the DVE approach, splitting a single UniPrep2 carousel loading into two distinctive runs is preferable. The first half of the carousel is loaded with samples and references for the initial low When ready for hydrogen-isotope analyses, the samples and references are transferred from the plastic storage plate into the UniPrep2 carousel wells and set up an appropriate CF-IRMS instrumental job list. An EA template comprises samples, references, and controls for the DVE and CE approaches. Example DVE and CE analysis templates for the UniPrep2 are found in Supplemental materials.The UniPrep2 carousel “zero” position should be manually aligned and verified to be slightly past the centre by approximately 1–2 mm, which ensures that open-top silver capsules do not lodge sideways in the EA opening. Manual alignments to the carousel must be made with the UniPrep2 power turned off to avoid damaging the electronic stepper motor. Before loading the samples, loosen the inner counter ring holding screw  and rotaStep 4. Setting UniPrep2 Equilibration and Drying Temperatureδ2Hn value. Some organic matrices may not be resilient to higher temperatures if coupled with a hot vapour exposure . In contrast, others, like hair, feathers, and chitin, are resilient to high temperatures >120 °C and vapour. Others, such as benzoic acid, may sublime, degrade, or change their structure with resulting hydrogen-isotope effects at lower temperatures. Regardless, the vapour equilibration step and the vacuum drying on the UniPrep2 should be conducted at the same temperature.Establishing a proper temperature for online static vapour equilibration and later drying on the UniPrep2 is essential for analysing each type of organic sample matrix. Generally, while hotter would be better (>40 to 90 °C) for achieving faster vapour equilibration, removal, and sample drying, the appropriate temperature will depend on the type of organic material being analysed for its Caution: There are no established rules for which equilibration temperature to use. For some complex organic matrices, the effect of heating in combination with vapour  and the impact on the δ2Hn value remain unknown δ2Hn results between 25 and 110 °C treatments using CE. Also, bear in mind that the boiling point of water is significantly reduced at low vapour equilibration pressures (approximately <5–10 mbar in the carousel). Conversely, residual water is always challenging to remove from metal and other surfaces without applying heat, vacuum, and helium flushing . Ultimately, a sound regimen for an organic sample matrix can only be successful and considered robust if δ2Hn determinations can be fully reproduced in another laboratory adopting identical standard operating procedures.2O injection port”. Turn on the UniPrep2 power switch (rear), if not already on, and then use the “Temperature Controller” on the control box to set it to the required vapour equilibration and drying temperature. Always use caution and wear heat-protective gloves when touching the hot surface of the UniPrep2 carousel. During the carousel heating, the samples can be evacuated to remove moisture and room air from the carousel. Turn on the membrane pump switch at the front of the control box and gently open the “Vacuum Out” valve on the UniPrep2 manifold. The gauge's pressure should drop and stabilize at <5 mbar. Continue evacuating the carousel and samples at the setpoint temperature and then continue to Step 5.After loading samples and reference silver microtubes into the carousel, close and tighten the carousel cover, ensure the “Isolation valve” and “He purge” valve is closed, and ensure a septum is in the “HStep 5. Conducting Static Vapour Equilibration for Hydrogen-Isotope Exchangeδ2H values that span the δ2H values of the samples. Ideally, the waters used for the static vapour equilibration are distilled or low in total dissolved solids to minimize salt buildup below the “H2O injection” port. However, minor salt residues can be wiped from the hydrophobic metal top inside the carousel. Generally, it is advisable to use two waters for vapour equilibration that are widely separated in their δ2HVSMOW values by at least 300 ‰ and span the expected δ2H range of the unknown samples. Their δ2H values should be well-established relative to the VSMOW-SLAP scale. These injection waters must be carefully stored in sealable glass bottles just like reference waters to minimize isotope fractionation by evaporation over time 2H enriched sample using laboratory-deionized water and gravimetrically add a precise amount of low-cost 99 % D2O to raise the δ2H value by >300 ‰. Establishing the δ2HVSMOW value of these vapour injection waters is critically essential (see below), but water isotope analysis is beyond the scope of this paper.All samples and references will need to be run twice for the DVE approach. Each sample set is statically equilibrated under heated vacuum conditions with two isotopically known reference waters having δ2H value of the equilibrating vapour does not matter (one may use laboratory-deionized water) because samples and references are all equilibrated with the same vapour and are normalized with the assigned δ-values of the matrix-equivalent reference materials, hence, use of distilled laboratory water for vapour CE equilibrations is convenient. Nevertheless, using water with a significantly higher or lower δ-value than the samples will allow for reliable detection of sample drying status, see below. Be aware that exƒ cannot be determined using the CE approach.For the CE method, the 2O injection port” septum is compromised, or the “Helium In” valve is not fully closed. . If the vacuum holds after closing the “Vacuum Out” valve (it should contain the vacuum indefinitely), turn off the “vacuum pump switch” on the front of the control box. The control box membrane pump is intended only for carousel pumping only and should never be left running continuously.After the samples and references have been loaded and the carousel top closed and sealed by tightening clamps, verify that the UniPrep2 “Isolation valve” is closed. Switch on the UniPrep2 power  and check that the “Temperature Controller” is at the correct equilibration temperature (40–90 °C). If using ambient room temperature for equilibration, set the “Temperature Controller” to 20–25 °C because the power must be on to activate the carousel (the controller defaults to 60 °C at start-up). Allow around 30–45 min for the temperature of the UniPrep2 carousel to stabilize. Ensure that the “He purge” valve on top of the carousel is closed, the “Helium In” valve is closed, and the 6.5 mm septum inside the injection port is sound. Turn on the “vacuum pump switch” in front of the control box and gently open the “Vacuum Out” valve to the carousel. Allow the carousel to be evacuated for approximately 30 min; the vacuum should be <5 mbar. After 30 min of pumping, close the “Vacuum Out” valve and observe the “Vac/pressure gauge” on the manifold – it should hold <5 mbar. If the pressure slowly or rapidly begins to rise, either the carousel top was not properly seated, the top “helium vent/in valve” is not fully closed, the “Hδ-value equilibration water) or CE, using a 100-µL glass syringe, obtain and then gently inject 30–100 µL (as required based on exƒ) of the low δ2H water (or DI water for CE) through the top septum injection port into the carousel. After the water injection, the pressure on the carousel gauge should slowly and visibly rise above 5 mbar, the degree of rise depending upon the carousel temperature and the amount of water injected. It will stabilize after approximately 3–5 min. A complete discussion of the amount of water needed for full vapour equilibration is found in 2O that can be injected into the UniPrep2 under vacuum conditions is < 200 µL; otherwise, there is the risk of a liquid water phase condensing inside the carousel. Allow the samples to statically equilibrate with the injected water vapour at the set temperature for at least 1 h to allow ample time for the samples and references to reach hydrogen-isotope equilibrium δ-value equilibration water the second time.Next, for DVE . Previous work has shown that 1–2 h vapour equilibration times are usually sufficient at elevated temperatures Step 6. Vacuum Pumping and Drying2O vapour inside the carousel must be removed. The samples are dried initially by evacuation and then by dynamic helium flushing to ensure complete drying. The vacuum pumping time depends on the samples' hygroscopicity and moisture retention capacity. As a starting point, the recommended vacuum time pumping is 30 min while monitoring the pressure gauge on the carousel manifold until it is steady at <5 mbar vacuum. Check to ensure the EA “Isolation valve” and “Helium In” valves are closed when vacuum drying. Turn on the “vacuum pump switch” in front of the control box. Gently open the “Vacuum Out” valve to ensure that powdered samples do not explode out of their capsules. Evacuate for 30 (or more) minutes. After evacuating, close the “Vacuum Out” valve. All the UniPrep2 manifold valves should be closed, and the carousel should hold at <5 mbar pressure. It is not recommended to forgo the H2O evacuation step and skip directly to helium flushing because the purge capillary inside diameter is exceedingly small and could become blocked with condensing water vapour.Following the sample-vapour equilibration step, all HStep 7. Helium flushing and sample dryingThe helium flushing and drying step that follows the initial vapour removal by evacuation is the final and critical step to ensure that samples are thoroughly dried online. The helium pressure and the capillary length control the helium flushing rate exiting the “He purge” valve. Using the factory-supplied capillary, a control panel helium pressure of 0.8, 1.0, and 2.0 bar helium should give carousel flushing rates of approximately 80, 100, and 150 mL/min. As a starting point, is it recommended to use 100 mL/min (1 bar) to flush the carousel, mainly because this helium pressure is close to the operating pressure of the EA, thereby minimizing pressure changes to the EA and CF-IRMS when opening the “Isolation valve.” If higher or lower helium flushing rates are needed, increase or decrease the helium pressure with the needle valve (max is 2 bar). Do not forget to set the pressure back to the EA helium operating pressure when the drying is completed. If a higher helium flow than 150 mL/min is desired (and not exceeding 2 bar helium), the vent capillary can be shortened by a few cm and retested for a faster flow rate.Set the helium pressure on the control box to 1 bar, gently open the “Helium In” valve on the manifold and allow the carousel pressure to stabilize. Next, open the “Helium purge” valve on the top of the carousel by rotating it counterclockwise by one complete turn (note: two turns will vent the carousel). This 1-turn setting allows pressurized helium to enter the carousel to pass over the samples and flow through the vent capillary at the set flow rate. If a digital flowmeter is available, check the helium flow rate at the end of the purge capillary and adjust using the helium needle valve on the UniPrep2 control box to get the desired flow rate. Carefully observe the outlet tip of the purge capillary for water droplets appearing after the first few minutes. With the initiation of the helium flushing, a few droplets may appear as the helium carrier gas effectively removes the remaining adhered water from the inside surfaces of the carousel and the samples.The time required for helium flushing can vary to remove all residual moisture and will need to be experimentally determined based on the sample matrix type and the hygroscopicity of the samples. For example, some complex organic samples like keratins may require <2 h of helium flushing, and other types of organics may require 4 h, or longer (next section).After the helium flushing and drying step is complete, check and readjust the helium pressure on the needle valve to match the EA and CF-IRMS system carrier pressure. Then tightly close the “He purge” valve (clockwise rotation). All valves on the UniPrep2 should be closed, and the carousel is now pressurized with helium. The samples are now ready for hydrogen-isotope analyses after a dryness check. As one final check before drying and H isotope analysis, using a handheld helium leak detector, check all of the UniPrep2 fittings, connections, and valves; the EA system should be helium leak-free.2 gas; however, this would require the user to add some gas fittings and valves to allow switching between N2 and He, bearing in mind that helium is the more effective vapour carrier). – this is typically <20–40 mV H2 signal and should be at once stable.CF-IRMS background: IRMS is •opened to the EA-HTC reactor (no sample dilution on the Conflo) – this background is typically higher but <120–300 mV depending on the type of reactor, its operating temperature, and IRMS tuning. After opening the EA reactor to the CF-IRMS, it can take 30 min or longer for the system to stabilize and reach a stable “EA background.” The best approach is to set up a time scan  and monitor the H2 signal over time. Typically, a low and stable EA background is achieved in <15–30 min.CF-IRMS background: IRMS •2 signal (see below): Carousel opened to EA and CF-IRMS.The UniPrep2 background or dryness HBefore starting the hydrogen-isotope analyses, a sample dryness check using the CF-IRMS is required until a routine drying protocol is determined. After opening the UniPrep2 sample carousel to the EA reactor, any residual H2 signal above the EA background. (Note: an empty UniPrep2 carousel looks like an EA background regardless of the carousel temperature). The UniPrep2 background test applies to both CE and DVE approaches. Any residual moisture emanating from the carousel or the samples will be carried through the EA-HTC and raise the H2 background, thereby quantitatively revealing insufficient drying. This dryness check should be performed as follows:•2 background in mV .With the UniPrep2 “Isolation valve” closed, check the EA-IRMS H•2 .Start an IRMS time scan for H•Gently open the “Isolation valve” on the UniPrep2 to the EA-HTC reactor.•2 trace on the IRMS time scan. Usually, there are minor bumps or an H2 pulse due to the EA valve opening and helium pressure changes. Then the UniPrep2 background should decrease to approximately 120–200 mV and stabilize after approximately 10–15 min. If the H2 background is acceptably low (<200 mV) and stable beyond 30 min, it can be deduced that the samples are dried, and hydrogen-isotope analyses can proceed.Observe the H•2 background will rise . It may rise well above 1000 mV, then slowly drop over time as H2O is carried from the carousel through the reactor. In this case, additional vacuum and helium flushing is required to dry the samples and carousel .However, if residual moisture is in the carousel or samples, the HTo assess the dryness of the samples, the UniPrep2 is opened to the HTC reactor and IRMS and is observed for an unexpected rise in H2O background as residual moisture remains in the samples. For example, after only 10 min evacuation pumping and 15 min of helium flushing on a carousel of keratin samples, opening the carousel to the EA reactor led to a rise in the UniPrep2 background on the IRMS to >2000–3000 mV – clearly, there was still considerable residual moisture still to be removed. After 3 h of helium flushing, the carousel was stable at UniPrepUniPrep2 background of approximately 120–160 mV. Importantly, it is likely that evacuation and helium flushing-time procedures will be sample-matrix specific. Hence, the researcher must determine the helium drying times for each organic sample matrix type.Experience has shown that insufficient evacuation and helium flush times after sample vapour equilibration will lead to considerable H2H that is not close to that of the sample), and after 30 min of evacuation, helium flush the carousel for 1 h, then analyse the first triplicate set, then flush the carousel with helium for another hour, analyse the second triplicate set, and so on spanning for as many hours as needed. The δ2H values of the samples should reach an asymptote after a few hours or more, revealing the minimum online drying time. The samples are deemed thoroughly dried if there are no further changes in the δ2H values of the sample over time and helium flushing. This δ2H asymptote in hours (and perhaps adding 0.5 h as a precaution) should be applied to samples of that matrix type. Hydrophilic organic samples are expected to take considerably longer to dry online, with helium flush times possibly exceeding 3–4 h compared to less complicated organics . With more UniPrep2 drying experience in various laboratories, it is expected that a standardized helium (or dry N2) drying schema could eventually be established for routine application to the most common organics.The procedure to determine the helium flush time needed to achieve dried samples is as follows: load the UniPrep2 carousel starting with a set of silver microtube reference waters  and then several sequences of triplicates of one matrix equivalent unknown sample. After vapour equilibration of the entire set of samples . With the chromium reactor configuration, and if fitted with a TCD, a small N2 peak may follow the H2 peak for nitrogenous samples on the TCD trace. With the carbon reactor configuration, a variable-sized N2 and a significant CO peak appear on the TCD after approximately 300–500 s. Again, all these timings are CF-IRMS and EA-specific and should be adjusted to ensure a complete return to EA baseline between samples. The H2 run should be checked for H2 retention time, peak shape, and peak area. Care should be taken that the EA reactor does not plug up with silver capsules, which generally leads to H2 peak broadening and an increase in retention time. Process the samples, standards, and controls as per manufacturer IRMS software or LIMS for Light Stable Isotopes (https://water.usgs.gov/water-resources/software/RSIL-LIMS/), and process the samples to report their final δ2H values on the VSMOW-SLAP scale.With the samples proven to be fully dried online and the UniPrep2 background stable, the samples are ready for δ2Hn CalculationsStep 10. ex) of the organic unknowns, the mass fraction hydrogen, and the δ2Hn. The ƒex result is obtained from measured δ2H isotopic difference in the unknown organic samples after equilibration with each of the two known and widely differing water vapours. For example, two vapours having δ2H values of +1.83 and −397.7 ‰ (VSMOW) were equilibrated with one unknown organic keratin sample. Using these two waters, the measured  δ2HVSMOW values of the organic sample after each isothermal vapour equilibration were −80.0 ‰ and −91.6 ‰ (VSMOW), respectively. To determine the fraction of exchangeable hydrogen:With the DVE approach, vapour equilibration of samples using two isotopically differing waters allows for the determination of the fraction of exchangeable hydrogen (ƒex = (δ2Hstd‐EW – δ2Hstd−DW) / (δ2Hw‐EW – δ2Hw‐DW)ƒex = (10.63 ‰ / 399.5 ‰) = 0.027 (or 2.7 % exchangeable hydrogen)ƒδ2Hn result is determined using either the high δ or lower δ vapour-equilibrated sample after an accurate ƒex has been determined:The δ2Hn = (δ2Hsample - (ƒex * (δ2Hw‐EW – δ2Hw‐DW)) / (1 - ƒex)δ2Hn = (−91.6 – (0.027 * 399.5) / (1 – 0.027) = −83.3 ‰ (VSMOW) where δ2H and EW and DW subscripts refer to the measured organic δ2H results  for the 2H-enriched and 2H-depleted value vapour exposures. In previous publications, the δ2Hn determination might have included an extra hydrogen-isotope fractionation factor (εsample-vapour). However, this isotope-fractionation factor can only be determined if organic samples can be chemically treated to remove the exchangeable hydrogen fraction  and remeasured for the δ2H difference after vapour equilibration. Because most complex organic samples cannot be chemically treated by nitration, it is recommended to deprecate the incorporation of a temperature-dependant vapour-sample hydrogen-isotope fractionation factor for most organics ex is crucial because it controls the outcome and the accuracy of δ2Hn. If a sample has no exchangeable hydrogen, ƒex should be zero . If an organic sample is dried, ƒex should produce the same value regardless of the isotopic composition of the two equilibration vapours used. However, suppose the ƒex of a sample differs when using a separate set of 2H-enriched or 2H-depleted vapours. In that case, this could result from incomplete drying and lingering residual moisture in the samples (or the carousel). Quantifiable drying using the IRMS as the detector ensures that residual moisture is not a confounding factor  and accordingly could lead to δ2Hn differences produced amongst laboratories if not repeatable. The ƒex may be an additional useful diagnostic feature of the organic sample type in addition to its δ2Hn value. Generally, fresh organic matter and carbohydrates have higher ƒex values, whereas refractory organic matter usually has a lower ƒex.The determined ƒ2 peak areas and hydrogen mass fractions to the sample peak areas and thereby calculating the fraction of hydrogen of the samples using either the CF-IRMS H2 peak areas in LIMS for Light Stable Isotopes, or, if an EA TCD option is available using the H2 peak areas and conducting offline calculations. The elemental hydrogen composition of an organic sample should be stable and consistent after online drying. For example, suppose an organic reference sample  with a certified 6 % total hydrogen mass fraction gives a determination with a higher value . In that case, it is likely caused by residual moisture or from sample weighing errors. Careful drying and accurate sample and reference weighing are critical for mass fraction determinations.The elemental H mass fraction of the organic samples should also be determined and reported after CF-IRMS analysis. The mass % of H must be closely monitored with the DVE approach because this assay is also impacted by residual moisture that is measured as part of the total hydrogen. The elemental hydrogen mass fraction can be determined for unknown samples by (i) including accurately weighed samples of non-exchangeable H reference materials in each run with established elemental hydrogen concentrations  and( ii) comparing their Hδ2Hn uses a two-point isotopic normalization using the δ2Hn organic reference materials having consensus values on the VSMOW-SLAP scale. Single-point normalization is not recommended due to IRMS-specific scale expansion or contraction. Because so few δ2Hn working references exist, a three-point or more-point scale normalization is unlikely to be available.Using the CE approach, the determination of δ2Hn sample) is done using the CBS (δ2Hn = −157 ‰ VSMOW) and KHS (δ2Hn = −35.3 ‰ VSMOW) keratin standard references In this example, a two-point normalization δ2Hn hair = δ2Hkeratin std low VSMOW + (δ2Hhair meas -δ2Hkeratin std low meas) x ((δ2Hkeratin std high VSMOW -δ2Hkeratin std low VSMOW) / (δ2Hkeratin std high meas -δ2Hkeratin std low meas))δ2H values for CBS and KHS from the CF-IRMS against H2 tank reference gas were +53.7 ‰ and +203.9 ‰ for CBS and KHS, respectively. The measured δ2H IRMS value for the hair sample was +99.3 ‰. Using the above equation, one can determine the δ2Hn of the hair sample (and those of others in the run):In the laboratory, the uncorrected δ2Hn hair = −157.0 + (99.3 - 53.7) x ((−35.3 - (−157)) / (203.9 – 53.7)) = −120.1 ‰ VSMOWδ-value to δ2Hn on the VSMOW-SLAP scale. Notably, this normalization does not include other pre-processing data corrections that could be applied to improve the precision of the results, like ion source linearity corrections to correct for a predictable isotopic variance from sample weighing or time-linear instrumental drift, which are both readily determined and applied using data processing software like LIMS for Light Stable Isotopes.The 2-point normalization converts the uncorrected hair δ2Hn values for organic working standards from older literature or non-authoritative sources. The status of organic δ2Hn reference materials is still unsettled, and the δ-values of some organic standards have changed considerably over time as hydrogen-isotope analysis methods. The recognition of the impact of exchange and drying has also evolved. Practitioners should be vigilant to the δ-values used by the laboratory for its organic references and report these in their publications. Only with full disclosure of operational conditions and assigned values can the results be used by others for comparative studies, for example, in an extensive exercise in the recalibration of hydrogen-isotope results obtained from multiple laboratories over several decades Caution should be used when quoting After the organic hydrogen samples have been successfully analysed using the UniPrep2, close the “Isolation valve" to the EA and CF-IRMS, turn off the UniPrep2 power, and let the carousel cool to room temperature. As required, close the EA to the CF-IRMS system on the mass spectrometer software. Ensure the “Vacuum Out” and “He in” valves are closed. Open the “He purge” valve on the top of the carousel (two counterclockwise rotations to vent) and allow the carousel to release its pressurized helium contents to atmospheric pressure as indicated by the Pressure gauge, and then firmly close the top “He purge” valve again. Loosen the carousel wingnuts and open the hinged cover plate. Check each sample well for silver capsules that may have gotten stuck and for signs of spilled sample powder or particle residues that could contaminate subsequent analyses. The carousel can be cleaned by wiping the top with a lint-free cloth and the sample wells by gently blowing them with compressed canned air. Loosen and reset the sample counter ring to the correct zero position. Depending on the number of samples and the amount of silver buildup, it may be needed to cool the EA, stop the helium flow, and remove and clean out the reactor of the silver capsule buildup. Unscrew and detach the UniPrep2 adaptor from the EA flange and then gently move it to the side or back to allow reactor removal, and take care not to damage the helium carrier gas lines or fittings. Follow the EA manufacturer instructions for HTC reactor removal and reinstallation. With a newly cleaned reactor in place, firmly re-attach the UniPrep2 to the EA reactor flange in readiness for equilibrating and running the next set of samples. It is recommended to close and evacuate the UniPrep2 carousel when not being used to avoid external dust from getting into the sample wells.δ2Hn values, method validation is only possible using several USGS organic reference materials, for example, using the CBS and KHS keratins as the high and low bracketed calibration standards (either CE or DVE as described above) and running the USGS42 and USGS43 hair keratins as unknowns. Either approach should return the USGS-certified hair VSMOW δ values, as demonstrated in δ2Hn reference materials eventually become available, standard operating procedures (SOP) validation and interlaboratory replication are envisioned to play a vital role in advancing hydrogen-isotope analysis for different organic environmental matrices and studies.Given the lack of diverse organic reference materials for No ethical considerations were required.Leonard I. Wassenaar: Conceptualization, Methodology, Writing – original draft, Validation. Leonardo Sisti: Conceptualization, Visualization, Writing – review & editing. Matthias Pilecky: Writing – review & editing. Martin Kainz: Resources, Writing – review & editing.The authors declare that they have no known competing monetary interests or personal relationships that could have appeared to influence the work reported in this paper.


Our simulations show synergisticeffects due to the presence of PAHs compounded by ion-specific effects.Our analysis reveals phenomenological correlations between the wettingproperties and a combination of the binding free-energy differenceand entropy changes upon oil solvation for PAHs at oil/brine and oil/hydrateinterfaces. The detailed thermodynamic analysis conducted upon theinteractions between PAHs and various interfaces identifies molecular-levelmechanisms responsible for wettability alterations, which could beapplicable for advancing applications in optics, microfluidics, biotechnology,medicine, as well as hydrate management.Polycyclic aromatic hydrocarbons (PAHs) have attractedremarkablemultidisciplinary attention due to their intriguing π–πstacking configurations, showing enormous opportunity for their usein a variety of advanced applications. To secure progress, detailedknowledge on PAHs’ interfacial properties is required. Employingmolecular dynamics, we probe the wetting properties of brine droplets(KCl, NaCl, and CaCl To fill this knowledge gap, we implementatomistic molecular dynamics (MD) simulations to examine the wettingbehavior of brine droplets  on sIImethane–ethane (C1–C2) hydrates coated with PAHs andimmersed in oil solvents. The simulation results show significantdifferences in wetting properties observed for different brine droplets.Inspired by Prausnitz’s commentary33 in reference to the well-known book by Lewis and Randall,34 we invoke the “broad highwayof thermodynamics” to explain the new phenomena ofhydrate wettability observed here, with the ambition of pioneeringa growing field. In particular, we attempt to explain differencesin wetting behavior observed as a function of salt and solvent typeby conducting advanced thermodynamic analyses, including binding free-energyand configurational entropy changes upon solvation.Promoting advances in technologies such as those listed aboverequiresa better understanding of interfacial properties in the presence ofPAHs. Hence, in this work we quantify the effect of self-assembledPAH structures on the wettability of clathrate hydrates, systems whichhave significant direct relevance for preventing the formation andagglomeration of hydrate particles with brine droplets.X, Y, and Z dimensions of 10.386, 5.193, and 1.731 nm, respectively, followingour previously published procedures.35 More detailson the construction of hydrate substrates are reported in the Supporting Information (SI). Then, we placed28 PAH molecules on top of the hydrate surface, and we equilibratedthe system at 274 K and 3.45 MPa until equilibrium. Equilibrium wasconsidered achieved when the system energy as well as density profilesof solvents along the Z direction perpendicular tothe hydrate surface converge. We present the 2D density profiles obtainedfor PAHs adsorbed on the hydrate surface in In 2 cylindrical droplets ,36 periodicalong the Y direction, were situated on top of thesolid substrate. The simulation box was then filled with organic solvents,prepared by varying the heptane–toluene relative composition when the solvent is pure toluene, inwhichcase our simulations, in some circumstances, reveal spontaneous rollingof the brine droplets on the PAH-covered hydrate surface (see detailsin Movie S1 in the Supporting Information).In this latter case, the contact angle was estimated when the dropletresided in one position at the PAH-covered surface 8(H2O)4 with an inner shell of four water molecules and anouter layer of eight benzene molecules, while Williams et al.43 reported that six water molecules provide anadequate shielding that prevents benzene from closely interactingwith Ca2+. These self-assembled clusters in effect leadto an aqueous–aromatic phase separation at the nanoscale.43 Similar phenomena could explain why NaCl andCaCl2 droplets located on the PAH-covered hydrate maintaintheir cylindrical shape in pure toluene, while the KCl droplet graduallypenetrates the adsorbed PAH–toluene film, reaching the hydratesurface, and ultimately yielding a contact angle of ∼90°.Our resultscomplement literature reports. For example, investigatingion effects on surfaces covered by self-assembled monolayers terminatedwith benzene, Rimmen et al.+ and Ca2+ ions (contact angle of ∼36°) . Similarly,we also observe the negligible differences in the contact angles obtainedfor water droplets on the hydrate surface covered with PAHs in theabsence of salt ions .To put these results in perspective, it helps observing that, whenPAHs are not present, our simulations reveal insignificant differencesin the contact angles obtained for brine droplets on the bare hydratesurface immersed in solvents, even pure toluene, for which an aqueous–aromaticphase separation would be expected in the presence of NaOne fundamental question arisesfrom the quantitative analysisabove: what mechanisms are responsible for the remarkable differencesin the wetting behaviors of the brine droplets shown in 2 droplets on the hydrate surfaces are similar for all solvents simulated.In imulated2. The PM47 were conducted. This method allows us to estimate the free-energy(FE) landscape sampled as the orientational angle formed between thevector normal to the PAH polyaromatic core and the surface normalchanges along the Z direction (details provided inthe SI). In Figure S3, the FE landscapes show that the most stable structure ofthe PAH at the heptane–brine interface (blue) maintains thepolyaromatic PAH core nearly parallel to the interface (∼17°)while the PAH preferentially orients its polyaromatic core perpendicularto the heptol50/brine (red) and toluene/brine (green) interfaces.It is known that the π–OH interaction between aromaticcompounds and water is attractive 48 and that this interaction becomes even moreattractive when K+/Na+/Ca2+ and Cl– ions are present.41 Theseeffects explain why we observe mainly parallel orientations of thepolyaromatic PAH core at the pure heptane/brine interface. Increasingtoluene content in the solvent seems to cause a competition betweentoluene and PAH for access to the interface, resulting in the preferentialperpendicular orientation of the polyaromatic PAH core when heptol50or pure toluene are used. This could explain why PAH experiences amuch stronger attraction to the pure heptane–brine interface(blue) compared to the other interfaces considered here.In an attempt to identify molecular features thatcorrelate withthese results, well-tempered metadynamics simulations49 and employed the weighted histogram analysis method50 to examine the interactions between PAH andsolvent–hydrate interfaces . This is possibly due to the negligible π–OHinteractions between PAH and the water molecules contained in theclathrate hydrates structures, which assemble into crystalline polyhedralcages stabilized by hydrogen bonds. Quantitative analysis of the PAHorientation at the solvent–hydrate interface reveals that thePAH preferentially orients its polyaromatic core closer to the toluene–hydrateinterface (∼59°) compared to other interfaces (∼76°),suggesting stronger attractions between the PAH and the toluene–hydrateinterface. The PAH–heptane/hydrate interactions are more attractivethan the PAH–heptol50/hydrate ones probably because of thefavorable π–π interactions between PAH and toluenein the bulk phase. It is worth noting that while the effective attractionbetween PAH and pure heptane/brine interface is comparable to theone between PAH and the pure heptane–hydrate interface, theattraction between PAH and solvent–hydrate interfaces is muchstronger compared to the corresponding ones at the solvent–brineinterfaces when toluene is present.Wealso conducted umbrella sampling simulationsaces see 3B. The races see 3A. Via vΔG)for one PAH molecule at the interface as the difference of free energiesin the bound and unbound states (see details in the SI):51φi is the PMF value associatedwith the ith bin along the distance l. The relative binding free-energy difference (ΔΔG) for the PAH–solvent/hydrate and the PAH–solvent/brineinterfaces is then calculated asTo gain a quantitative understandingof the interactions betweenPAHs and solvent–brine or solvent–hydrate interfaces,we calculated the binding free energy (ΔΔG obtained for the varioussystems considered. We observe a direct relationship between ΔΔG (columns) and contact angle (filled circles)as the toluene volume fraction increases for NaCl (red) and KCl (blue)droplets; specifically, the contact angle increases as the ΔΔG decreases. However, because the contactangle of the NaCl droplet is much larger than that of the KCl dropleteven though the ΔΔG obtained for thepure toluene/NaCl system is larger, we conclude that the ΔΔG alone cannot explain changes in clathrate hydrate wettability forthe systems considered here.In 54 we computed the configurationalentropy, SL, of PAH in solvents, at solvent–hydrateand at solvent–brine interfaces. We implemented the methoddeveloped by Schlitter and others,58 according to whom SL is quantified by the covariance of the Cartesian coordinatesof atoms of one PAH molecule via52Because of the significant entropiccontribution in modulatingthe solvation of complex molecules,ΔS = SL(ρ) – SL(ρ = 0). We report the sum of the entropychange TΔSOH + TΔSOW (columns in TΔSOH + TΔSOW (columns) and contact angles (filled circles) for NaCl droplets(red); i.e., the contact angle increases as TΔSOH + TΔSOW decreases.Notwithstanding these notes, a correlation is not observed for KCldroplets (blue).The entropy changebecause of the reorganization of PAH in thepresence of solvent at density ρ can be estimated as ΔΔG + TΔSOH + TΔSOW), we observea direct correlation for all systems considered; i.e., the lower thevalue of ΔΔG + TΔSOH + TΔSOW (columns),the higher the contact angle (filled circles)  see 3E, left.60 proposed that asphaltene moleculesconsisting primarily of one PAH (“island”) or multiplePAHs (“archipelago”), as observed by atomic force microscopyand scanning tunnelling microscopy,18 adsorb“flat on” the solvent–water interface as a monomer,while nanoaggregates do not adsorb as readily because of the protrudingaliphatic chains. These propositions, however, seem to be at oddswith several other studies, which invoke the importance of strongπ–π stacking interactions between PAHs.61 For example, Schneider et al.14 showed that hexahexabenzocoronene(HPAHBC) orients its PAH core parallel (∼10°) to the watersurface. As the number of HPAHBC molecules on the water surface increases,the aggregates form and adopt a parallel π–π stackingwith the PAH core plane forming a much larger tilt angle (90°).The results obtained for the systems of PAH aggregates  and the ones of PAH monomer in aromaticsolvents  considered inthe present work seem consistent with the latter observations. Althoughmore extensive data sets should be used to test whether the correlationproposed here has predictive capabilities, in ΔΔG + TΔSOH + TΔSOW for two PAH molecules that aggregated yielding a PAHdimer. The results were obtained for NaCl (yellow) and KCl (blue)brines and various solvents. We find it promising that a correlationsimilar to the one obtained for individual PAH molecules exists alsobetween ΔΔG + TΔSOH + TΔSOW for the PAHdimer and the brine droplet contact angles.Because inmany applications PAHs are expected to agglomerate,one could question whether the promising results presented in ates see S4 and th2 brine droplets wasobserved on PAH-covered hydrate surfaces (the interested reader isreferred to Movie S1 reported in the SI).More importantly, this study reveals a direct correlation betweenthe wetting properties and a combination of binding free-energy differenceand entropy changes upon solvation for PAHs at solvent–brineand solvent–hydrate interfaces. While this correlation holdsfor the systems considered in this work, more extensive studies, conductedfor a variety of salts and solvents, should be conducted, perhapsguided by a design of experiments approach, to assess whether thiscorrelation is general. Our results emphasize the importance of molecularthermodynamics in explaining the molecular mechanisms governing wettabilityalteration, phenomena that are of importance for further developingapplications of PAHs in optical, microfluidics, manufacturing technologies,as well as in hydrate management.In conclusion, oursimulations demonstrate that the interplay ofPAHs, aromatic content, and salt-specific effects impacts significantlythe interfacial properties of PAHs, and specifically their abilityto influence the wettability of clathrate hydrates. In some cases,spontaneous rolling of NaCl and CaCl


Only 5% of AD cases are familial making it difficult to predict who will develop the disease thereby hindering our ability to treat the causes of the disease. A large population who almost certainly will, are those with Down syndrome (DS), who have a 90% lifetime incidence of AD. DS is caused by trisomy of chromosome 21 resulting in three copies of APP and other AD-associated genes, like dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) overexpression. This implies that DYRK1a inhibitors may have therapeutic potential for DS and AD, however It is not clear how overexpression of each of these genes contributes to the pathology of each disease as well as how effective a DYRK1A inhibitor would be at suppressing any of these. To address this knowledge gap, we used Drosophila models with human Tau, human amyloid-β or fly DYRK1A (minibrain (mnb)) neuronal overexpression resulting in photoreceptor neuron degeneration, premature death, decreased locomotion, sleep and memory loss. DYRK1A small molecule Type 1 kinase inhibitors (DYR219 and DYR533) were effective at suppressing these disease relevant phenotypes confirming their therapeutic potential.Alzheimer’s disease (AD) involves pathological processing of  There remains no cure or treatments for the causes of AD, with only a limited number of drugs that treat some symptoms in some people. Cholinergic neuron loss in the hippocampus and cortex is prominent, leading to compromised memory, with extensive neurodegeneration finally leading to premature death3. AD is also associated with disrupted circadian rhythms and sleep8. Post-mortem slices allow staining of extracellular plaques of amyloid-β (Aβ) consisting of neurotoxic Aβ peptides such as the 42 amino acid (aa) peptide (Aβ42). Aβ42 is the product of amyloidogenic cleavage of amyloid precursor protein (APP) by secretases. Mutations in APP and within some of these secretases are a cause of familial AD9. The synapse loss and cognitive decline evident later in AD most strongly correlate with the accumulation of intracellular tangles of hyperphosphorylated Tau protein. Tau is phosphorylated by multiple kinases, including GSK-3β, CDK5, JNK, MAPK, CaMKII and DYRK1A11. Tau is the product of the microtubule associated protein tau (MAPT) gene, which exists as six isoforms with variable numbers of N-terminal domains  and C-terminal tubulin binding repeats (e.g. 3R or 4R). Therefore, the reason why 4R Tau is more strongly associated with AD is that it contains four self-aggregating repeats compared to three in the 3R isoform making it more prone to aggregate and cause AD pathology13.AD is the most prevalent form of dementia with 42.3 million people with AD worldwide in 2020. With ageing populations, this number set to increase further, along with the economic costs that are already estimated to be $818 billionAPP, PSEN1 and PSEN2 causative mutations, with APOEε4 allele being the main genetic risk factor for sporadic AD14. This has also obstructed the prediction of who and when people will get AD, with diagnosis often only being confirmed post-mortem or after irretrievable loss of neurons, hence frustrating attempts at early intervention and development of causal treatments16. Most of our understanding of the mechanisms that underly AD pathology are limited to rodent models of familial AD based on misexpression of different APP or Tau transgenes that do not recapitulate progressive neurodegeneration and all of the behavioral hall marks of the disease. These genetic models may not fully recapitulate ~ 95% of the causes of AD, which are sporadic. This could partially explain the lack of translation of drugs that are effective in these models to clinical efficacy18. Therefore, there is a need for more diverse animal models that faithfully represent the pathology of both familial and sporadic causes of the disease and that allow high-throughput screening in a genetically and experimentally tractable system.About 5% of AD are early-onset familial forms due to autosomal dominant inheritance of Drosophila, holds some potential in this regard with neuronal overexpression of different human APP and Tau products leading to photoreceptor neuron degeneration, shortened lifespan and deficits in neuronal excitability, movement, circadian rhythm, sleep and memory28.29. DS is a syndrome involving complex developmental abnormalities leading to compromised motor control, learning difficulties and sleep disruption32. People with DS develop Alzheimer’s disease (AD)-like (AD-DS) pathology of Aβ plaques and accumulation of tangles of hyperphosphorylated Tau leading to neurodegeneration in people as young as 20 years old, causing progressive motor, cognitive and health decline in early middle age34 and, finally, shortening of life by ~ 30 years35. Therefore, overexpression of chromosome 21 genes may not only cause DS but also be an early driver of AD pathology. One such candidate gene is DYRK1A, which is highly expressed throughout the brain and further so in DS37, contributing to motor and cognitive deficits associated with the disease40. DYRK1A phosphorylates Tau at S396, T231, and S262 as well as changes the splicing of Tau isoforms, resulting in increased neurofibrillary tangle formation47. DYRK1A phosphorylates APP and promotes neurotoxic Aβ peptide processing39. Thus, DYRK1A impacts both major pathology pathways of AD.One large (1:450–1:2200 live births) cohort of people that can be predicted to develop AD (90% lifetime incidence) and which can be diagnosed in utero and then followed from birth are those with Down syndrome (DS) or trisomy 21. DS is caused by expression of three copies of chromosome 21 genes, including overexpression of APP and hence increased AD pathologyDYRK1A is mnb, that exists in five isoforms, E-I, which all contain the highly conserved kinase domain48. Mnb is a presynaptic protein, mutations in which disrupt growth of presynaptic boutons and recycling of transmitter vesicles. Expression of mnb-F rescues these defects49. Neuronal expression of three copies of the longest isoform mnb-H (1.5 × wildtype levels) increased the number of glutamatergic boutons, enhanced spontaneous vesicular transmitter release, and slowed recovery from short-term depression resulting in motor impairment that persisted in aged animals. These animals also displayed progressive neurodegeneration and shortened lifespan50.The fly ortholog of 45. The DYRK1A benzimidazole inhibitor DYR219 decreased Aβ42 aggregation and Tau phosphorylation, reversing cognitive deficits in 3xTg-AD mice that overexpress mutant APP K670M/N671L, MAPT P301L and Presenilin M146V51. The DYRK1A inhibitor SM07883 also reduced Tau hyperphosphorylation in mice overexpressing MAPT P301L, leading to decreased Tau aggregation, neurodegeneration and improvements in behavioural deficits44. Leucettine inhibitor (L41) treatment of DYRK1A overexpressing mice ameliorated brain connectivity and synaptic protein expression, rescuing cognitive deficits52. The drug CX-4945 is a competitive inhibitor of the ATP-binding site of DYRK1A which is required for its kinase activity, hence was able to correct the hyperphosphorylation of Tau, APP and PS1 caused by DYRK1A overexpression in cell lines47. In Drosophila CX-4945 decreased fly eye degeneration caused by human Tau overexpression and delayed lethality resulting from pan-neuronal overexpression of mnb-H47. Likewise, in mice overexpressing DYRK1A, CX-4945 decreased Tau hyperphosphorylation47. In addition, some of these drugs may also induce protein degradation of DYRK1A or movement of APP to lysosomes for degradation, thereby providing additional means to correct DS and AD pathology51. Therefore, DYRK1A inhibitors hold great promise for the treatment of DS and AD pathology. To characterise the novel DYRK1A inhibitors, DYR219 and DYR533, we compared the effects of overexpression of human Tau (0N4R), human tandem oligomerising secreted Aβ42 and fly mnb-H on degeneration of photoreceptor neurons, longevity, motor performance, sleep and memory measuring the ability of the drugs to suppress these DS and AD relevant phenotypes.Like AD there are no treatments for DS, with a lack of models and knowledge of underlying mechanisms hampering development of new drug targets and treatments. However, kinase inhibitors of DYRK1A kinase have shown efficacy in preclinical models of DS and ADD for DYRK1A of 16 nM and DYR533 which has a KD for DYRK1A of 1.4 nM have been described in two separate patents54. Based on the food concentration of DYR21954 that was previously shown to decrease Aβ42 and Tau pathology and to reverse cognitive deficits of AD mice, we fed control  or flies overexpressing AD associated human Tau 0N4R in all neurons (elav > Tau) 304 μM DYR219 and 248 μM DYR533. The UAS-human MAPT (TAU 0N4R)28, has been reported to be leaky55, however we found UAS-Tau 0N4R/ + controls were wildtype for many of the phenotypes reported here56, consistent with this not being a concern. Feeding these concentrations of drugs throughout development and adulthood produced viable offspring. Total protein was extracted from whole heads from control and human Tau expressing flies treated with the different drugs or vehicle and was equal in quantity in all lanes . DYR treatment did not cause a visible change in the level of total unphosphorylated Tau. Quantification of intensity of bands from three Western blots showed the levels of phosphorylated Tau were indeed reduced at sites pT231 and pS396 with DYR219 treatment  and the fly orthologue of DYRK1A called minibrain (mnb) using the Glass multimer reporter  promoter50. Compared to control  flies  overexpression of human Tau (38% shorter life), Aβ42 (50%) and mnb (25%) reduced  flies are tapped to a bottom of a tube which elicits a reflex causing the flies to move away from gravity with about 75% of flies reaching the top of the tube within 10 secs  but did improve the locomotor deficits of all flies with pan neuronal overexpression of the different disease-associated genes, partially rescuing Aβ42 and mnb flies while fully rescuing Tau flies to a level indistinguishable from wildtype control  which count beam-crosses flies makes under different lighting conditions with sleep defined as more than five minutes inactivity in a 30 min time window with sleep mostly sleeping at night but also in the day when males often “siesta” during the afternoon21. Flies that overexpressed human Tau, Aβ42 and mnb throughout the clock (Timeless (tim)-Gal4 promoter) decreased total  overexpression of human Aβ42, Tau and mnb decreased 1 h memory  transgenic which was generated over 20 years ago28, however as mentioned earlier this UAS-Tau 0N4R line has been reported to be leaky i.e., shows expression independent of the Gal4 promoter55. We are satisfied that this is not a major concern because we have shown UAS-Tau 0N4R/ + controls are wildtype for many of the phenotypes we report here56. Therefore, any potential Gal4 independent expression of the line is not sufficient to cause a phenotype relevant to this study.We found DYR219 and DYR533 DYRK1A inhibitors suppressed degeneration caused by mnb, Tau and Aβ42 overexpression. We showed the DYRK1A inhibitors block mnb phosphorylation of human Tau at T231 and S396 for DYR219 and T231, S262 and S396 for DYR533. Thereby inhibiting the increased kinase activity caused by × 1.5 mnb overexpression but also the pathology caused by human Tau and Aβ42 we propose via decreasing endogenous mnb phosphorylation and pathological processing of these neurotoxic molecules. We used a very widely used 56 and which are consistent with those reported here. We19 and others58 have also found the effects of UAS-Tau 0N4R/ + are relatively consistent with other human UAS-Tau insertion lines (e.g. UAS-Tau 2N4R), again consistent with leakiness of this specific insert not being the main contributing factor to the phenotypes reported.We have also used the line multiple times and found it always gives a range of robust AD-related phenotypes50 we found overexpression of AD-associated human Tau, Aβ42 and mnb (in this order of neurotoxicity) caused a rough eye phenotype, loss of photoreceptor neurons and a concomitant decrease in the size of the eye. This extends previous reports of that DYRK1A causes neurodegeneration in mice41 which then leads to loss of locomotor and cognitive performance in AD and DS which culminates in shortened life that is directly correlated with this pathology59. Interestingly in contrast to DYR219, DYR533 appeared to be able to suppress human Tau and Aβ42 degeneration independent of mnb, suggesting it may be able to directly block pathology via another mechanism. DYR219 was particularly effective at suppressing mnb mediated degeneration, completely rescuing eye size to that of wildtype control caused by mnb overexpression. This provides pharmacological validation of DYRK1A as a DS and AD therapeutic in a range of preclinical animal models. We do not know the reason for the differences in phosphorylation and phenotypic suppressive effects of DYR219 and DYR533. It might be that subtle differences in their chemistry, differences in their affinities, the concentration used and differences in circulating doses of the drugs in the fly, with DYRK1A being dose dependent in mice and human, with too little or much activity causing pathology54. For instance the drugs appear to have differential affects at inhibiting phosphorylation at S262, which is close to the first microtubule binding site so can modulate microtubule binding affinity, therefore phosphorylation at this site may be particularly associated with pathology13. In addition, the drugs may have differing effects on the different phenotypes studied which resulted from promoter misexpression of the different genes in different neuronal populations which may result in different levels of pathological gene products, differences in selective vulnerability of the different types of neurons and access by the drugs to the neurons.Confirming previous studiesWe showed that lifelong exposure to the DYRK1A inhibitors had no detectable effect on lifespan of control, however they were found to be effective at suppressing the shortening of life caused by pan-neuronal overexpression of AD-associated Tau and Aβ42. We also found that although both drugs were able to suppress the human Tau and Aβ42, DYR533 as opposed to DYR219 could suppress shortened lifespan caused by mnb overexpression. Regardless DYRK1A inhibitors generally seem effective at suppressing the increased human Tau, Aβ42 and mnb senescence.2+ handling60, which maybe differentially effected by the DYRK1A inhibitors. Regardless the DYRK1A inhibitors were partially effective at treating the motor deficits of the disease models and DYR533 was even able to fully rescue the motor performance of pan-neuronal mnb overexpressing flies to a level indistinguishable to control.We also demonstrated that the neurotoxic effect of overexpression of Tau, Aβ42 and mnb had severe behavioural and cognitive consequences reversible by the drugs. We found locomotor performance was compromised in all the disease models, with the DYRK1A inhibitors suppressing motor deficits of all genotypes, except DYR533 was not able to reverse the effect of Aβ42. This may be due to differences in circulating dose and affinity of DYR533 which maybe insufficient to rescue Aβ42 mediated behavioural phenotypes. Alternatively perhaps the phenotypes cannot be completely explained by the degeneration of the neurons the neurotoxic genes are expressed in, for instance Aβ42 can also disrupt neuronal function in more subtle ways, such as disrupting neuronal excitability and Ca5. Our work complements previous work on the effect of clock expression of Tau and Aβ42 on circadian rhythms and sleep disruption in flies including Aβ42 and Tau sleep less during the day and night, while expression of tandem Aβ42 and Tau caused behavioural arrhythmia and sleep fragmentation62, and extends this to show that clock expression of mnb also causes sleep loss.Clock-wide overexpression of Tau, Aβ42 and mnb reduced sleep both in the day and night with DYR219 increasing the sleep of mnb overexpressors in a level indistinguishable from wildtype, whilst DYR533 increased the sleep of flies overexpressing Aβ42 sleep. Intriguingly when the total sleep was split into day and night sleep, DYR219 was shown to increase day and night sleep in mnb overexpressor flies, while DYR533 fully corrected the loss of nocturnal sleep in Aβ42. Furthermore, DYR219 was able to make mnb overexpressor flies able to sleep for longer episodes at night, consistent with the drug improving consolidation and alleviating sleep fragmentation. It should be noted that people with AD suffer from sleep fragmentation as well as reporting difficulty sleeping at night making them susceptible to nocturnal wandering8 and rodent models of AD64. Prior to neurodegeneration flies and mice overexpressing human 0N4R tau neuronal hyperexcitability and inability to sleep at night64. Sleep has also been shown to remove Aβ42 and tau from interstitial fluid in rodents and cerebrospinal fluid in humans with levels increasing with wake time and being removed at night with sleep deprivation further exasperating pathological leading to tau spreading65. Therefore, DYRK1A inhibitors have potential as correctors of DS and AD associated sleep problems thereby causing a predicted increase in Aβ42 and Tau clearance, slow pathology and thereby boast cognition65. This work also validates the use of Drosophila as a high throughput system to screen for novel DS and AD circadian and sleep drugs66. Therefore, DYRK1A inhibitors have potential to be tested as sleep correctors for DS that would likely also have beneficial effects on cognition and motor skills6. Likewise, the drugs may also be effective for sleep disruption in AD with the additional potential benefit of correcting sun-downing, when the patients’ symptoms, anxiety and confusion are greater in the evening. Similarly, the inhibitors may suppress AD nocturnal insomnia, the increase in nocturnal wandering and associated poorer cognitive measures the next day69.Our work is consistent with what is known in AD, which shows compromised clock function with post-mortem AD brain slices revealing extensive neurodegeneration of the mammalian clock, the suprachiasmatic nucleus (SCN) in human70. Because of this and the fact that learning and memory difficulties are hallmarks of DS and AD we tested the effect of mushroom body (the memory centre of the fly) overexpression of Tau, Aβ42 and mnb which reduced memory. DYR219 suppressed the memory loss caused by human Tau completely rescuing performance to wildtype levels while DYR533 suppressed human Aβ42 memory loss completely rescuing it to control levels. DYR219 also rescued fly mnb mediated memory loss. This suggests that DYRK1A inhibitors have potential as use as cognitive enhancers for DS and AD. We also performed sensory controls that showed that the drugs had no adverse effects on olfaction or response to reinforcement.It is known that robust sleep–wake cycles improve memory function and sleep is required for long term memory consolidationGMR-Gal4) compared to all neurons , clock neurons  and mushroom body memory neurons . Again, suggesting that different types of neurons may display selective vulnerability to the neurotoxic effects of the different disease associated gene, with expression of human AD genes being more detrimental than expression of fly mnb. Therefore, the more extreme phenotypes of the human AD genes might be more difficult to fully rescue with a DYRK1A inhibitor, than the more subtle defects caused by fly mnb overexpression, for which the drug was designed. Likewise, this reiterates that the drugs may have different effects or access to the different neurons the genes are expressed in for instance, the photoreceptor neurons are peripheral and sensory, while the later promoter lines express in possibly less drug accessible central neurons. Future work could be aimed at optimising the dose and timing and treatment for the different models and assays.Again, we found the two DYRK1A inhibitors herein showed differential benefits on the different phenotypes of the models tested, likely due to their differences in inhibition of mnb mediated phosphorylation and because the different transgenes could have different neurotoxic effects in the different types of neurons that they were expressed in e.g. eyes . Tim(27)-GAL4/CyO19 was a generous gift of Dr Ralf Stanewsky . The following strains were obtained from Bloomington Drosophila Stock Center : elav-Gal4/CyO (8765) OK107-Gal4 (854) and GMR-Gal4/CyO (9146). UAS-human MAPT (TAU 0N4R) wild-type was kindly given by Dr Linda Partridge, University College London)28, UAS-human tandem Aβ42-22 amino acid linker-Aβ4221  and UAS-mnb flies  were kindly provided by Dr Kweon Yu (Korea Research Institute of Bioscience and Biotechnology). Fly strains including Tim-Gal4, OK107-Gal4, elav-Gal4 and UAS-Tau that were routinely used for behaviours that might be affected by genetic background, were fully cantonised for this study, as described previously56, a study where we found cantonisation had little effect56 on many of the phenotypes reported here.Flies were raised on a standard corn yeast cornmeal diet  at 25 °C on 12 h light:12 h dark (LD) cycles and. D) of 16 nM) and DYR533 (KD = 1.4 nM) were prepared in multi-gram quantities54 and are available on request from Dr Christopher Hulme (hulme@pharmacy.arizona.edu) under MTA with University of Arizona. Dissociation constants were determined using the DiscoveRx KD Elect assay described at www.discoverx.com. Both compounds were evaluated for selectivity against 468 kinases via KinomeScan™ technology at 1 μM (DYR219 S(35)-selectivity was 0.19 compared to DYR533 S(35)-selectivity being 0.032). Based on the concentrations of DYR219 (and similar DYRK1A inhibitors) in food that were found to be effective at suppressing neurodegeneration and cognitive deficits of 3xTg-AD and other mice51 we dissolved 100 mg of DYR219 and DYR533 in aqueous solution and then mixed the drug solutions into cooling (~ 40 °C) liquid fly food producing a final concentration of 304 μM (DYR219) and 248 μM (DYR533). Flies laid onto the drug food with the offspring developing and feeding on the drug throughout their lives including during phenotypic testing.The small molecule DYRK1A inhibitors, DYR219 (with DYRK1A dissociation constant (KWhole fly heads of mixed sex flies were dissected and placed in RIPA Lysis Buffer (Thermo Scientific) with 1:100 Protease/Phosphatase Inhibitor Cocktail  and homogenized. After 15 min 12,000 rpm centrifugation at 4 °C, the supernatants were collected, and protein concentrations determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific).Protein samples were then prepared by adding 1:4 of 4 × Bolt™ LDS Sample Buffer (Thermo Scientific) and 1:10 of 10 × Bolt™ Sample Reducing Agent (Invitrogen) followed by heating at 95 °C for 3 min. Samples of 100 µg total protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using precast 4%-12% Bolt Mini gels were transferred to PVDF membrane (Thermom Scientific). The membranes were blocked overnight at 4 °C in blocking solution , 150 mM NaCl, with 0.1% Tween 20 [TBS with Tween (TBST)] and 5% [wt/vol] bovine serum albumin (BSA)) and incubated at 4 °C overnight with the primary antibody: mouse anti-Tau , mouse anti-β-actin , anti-Tau phospho- S262, S356, S396 or T231 (Abcam) (1:500) in TBST containing 1% BSA. After three washes with TBST, the blots were incubated for 1 h at room temperature with horseradish peroxidase–conjugated mouse or rabbit IgG secondary antibody  and then washed three times with TBST. Detection was performed using Western ECL Substrate (GE) according to the manufacturer's instructions and developed on X-ray films first and then scanned. The relative protein expression levels were quantified by densitometry using ImageJ Gel Analysis software. Western blots from at least three independent biological replicate experiments for each fly strain were used for quantification.Glass multimer reporter  promoter line. 2–5 day old female flies were CO2 anesthetised and ethanol immersed to euthanise the fly preventing movement during image capture71. A Zeiss SteREO Discovery.V8 stereomicroscope (with up to 8 × magnification) attached to a Zeiss AxioCam MRm camera was used to image and assess the qualitative degenerative phenotype of “rough eyes” and quantify surface area using Zeiss Zen software which was analysed with 2-way ANOVA with multiple comparisons tests.Overexpression of transgenes in the developing and adult photoreceptor neurons was driven by the 55. Mantel-Cox survival curves were plotted and log-rank tests performed to compare survival between genotypes and drug treatment groups.Five groups of 10 two days old-mated females  were housed in vials containing standard food with or without drug at 25 °C, with all the genotypes and treatments being performed in parallel. The sex and mating status of the fly affects lifespan therefore we standardize across groups by picking mated females. The number of deaths was counted appropriately every two days with the remaining flies transferred to a new food vial with or without drugselav crosses used for the survival assay. They were given ~ 1 h to acclimatise to a standard empty food vial at 25 °C. Flies were gently tapped to the bottom of the 7.5 cm plastic vial this initiated the negative geotaxis reflex allowing the number of flies that crossed a line drawn 2 cm from the top of the tube in 10 s to be counted. This was then expressed as a % referred to as the climbing performance with 2-way ANOVA with Dunnett’s multiple comparisons being employed to analyse data50.Five groups of 10 two-five days old male flies were collected for each genotype and drug treatment group. Although the sex of the fly does not influence climbing, to save animals we use the males from the progeny of the Drosophila Activity Monitoring (DAM) system  this allowed quantification of sleep based on activity data from five days of 12 h:12 h LD which is counted in one and 30 min bins. The sex of the fly does affect sleep, and we pick males as they do not produce progeny whose activity would confound activity monitoring. Sleep was defined as bouts of inactivity lasting more than five minutes as per convention72. The mean total sleep, mean sleep in the day and night and sleep episode length were calculated for each individual using the Sleep and Circadian Analysis MATLAB Program (SCAMP) in MATLAB19.Two-five days old male flies were put into single tubes in +) which was paired with twelve 70 V DC electric shocks  for one minute. The flies were then given 45 s rest of fresh air to clear the tube of CS+ before being exposed to the second odour (CS-) without being given any electric shock. The odorants used were either 3-octanol  or 4-methylcyclohexanol  diluted in 10 ml mineral oil and adjusted to a concentration (~ 1:1000) which was equally aversive to the flies. After a 1 h rest on food intermediate-term memory (ITM) was measures using a performance index (PI) which was calculated as follows:CS− and NCS+ is the number of flies choosing CS− and CS+ arms of a T maze. The CS+ odour was reversed in alternate groups of flies to minimise odour odorant with the mean of the performance between the two consecutive odour trials giving a n = 1 . The olfactory and shock reactivity of the different genotypes on and off drug were performed as sensory controls. For shock reactivity, flies were placed at the choice point of a T maze of two shock tubes, one of which delivered the shock protocol above. The number of flies avoiding the shock over the total number of flies in the trial was used to generate a % shock avoidance. Likewise, % avoidance of the concentration of the respective odours versus air was calculated.20–40  two-five days old flies were collected and acclimitised to LD 25 ºC and 70% relative humidity conditions. The olfactory shock conditioning assay was performed under dim red light, to which the flies cannot see allowing them to concentrate on odour cues. Flies were placed in the training tube lined with an electrifiable grid and given 90 s rest in a stream of fresh air, prior to exposure to the first dour  with normality checked for all datasets using the Shapiro–Wilk’s test, prior to selection of the appropriate parametric or non-parametric statistical test. The tests used and the number of animals (n) used for each dataset is given in the corresponding figure legend. Data is presented as Mean ± Standard error of the Mean (SEM) and statistical levels shown as following non-significant (ns) Supplementary Figures.