These FTEs were observed just upstream of the Earth's postnoon magnetopause by the National Aeronautics and Space Administration (NASA) Magnetospheric Multiscale (MMS) spacecraft constellation. The minor ion characteristics are described using energy spectrograms, flux distributions, and ion moments as the constellation encountered each FTE. In conjunction with electron data and magnetic field observations, such observations provide important contextual information on the formation, topologies, and evolution of FTEs. In particular, minor ions, when combined with the field‐aligned streaming of electrons, are reliable indicators of FTE topology. The observations are also placed (i) in context of the solar wind magnetic field configuration, (ii) the connection of the sampled flux tube to the ionosphere, and (iii) the location relative to the modeled reconnection line at the magnetopause. While protons and alpha particles were often depleted within the FTEs relative to the surrounding magnetosheath plasma, the He Long‐duration magnetosheath FTEs observed under at postnoon local times and under similar solar wind conditions are compared and contrastedMinor ions from the magnetosphere are observed at higher energies at the edges than at the center of the FTEsThe cores of the magnetosheath FTEs are on closed field lines Various models have been proposed. Russell and Elphic  suggesteFrom the time of initial identification of FTEs in International Sun‐Earth Explorer (ISEE) spacecraft magnetometer observations  within FTEs are used to understand the topology and processes related to this phenomenon. However, even with such information, it can be difficult to unambiguously determine the sources. For example, while the appearance of bidirectional streaming accelerated electrons associated with a flux rope or an FTE is a strong indicator of closed magnetic field lines, it is challenging to determine if these field lines are magnetospheric field lines wrapped about the flux rope, or if reconnection occurring in multiple locations has closed the field line  component, and nominal solar wind density and solar wind speeds). The remainder of this paper compares and contrasts in detail the characteristics of these cases, with focus on the minor ion composition.Using observations from the National Aeronautics and Space Administration (NASA)/Magnetospheric Multiscale (MMS) mission . A novel Radio Frequency (RF) system within the HPCA Electrostatic Analyzer is used to reduce the relatively large observed proton population by a known factor while allowing the full flux of minor ions to pass through the instrument and into the time‐of‐flight section of the instrument for mass discrimination  is included. Convected solar wind magnetic field observations from the Wind Magnetic Field Investigation (MFI) in Geocentric Solar Magnetospheric (GSM) coordinates  is such that only a small fraction of each orbit can be downlinked in full resolution burst mode. Burst mode intervals are selected by the “Scientist‐in‐the‐Loop” (SITL) for each orbit for downlink. Identified FTEs during the mission's dayside phase are usually ranked by the SITLs at a slightly lower priority than magnetopause crossings, and so (depending on buffer sizes and queue) not every FTE interval is brought down in burst mode; a few are instead brought down in fast survey mode at significantly reduced resolution. The identification of FTEs by the SITL was based primarily on magnetic field signatures ; associated accelerated flows (if present) were also often noted. While the SITLs have provided very useful comments describing their selection of interesting events as observed by MMS, it is important to independently verify candidate FTE encounters from the observations.FTEs typically pass over sampling spacecraft in a few tens of seconds, and the MMS HPCA instruments observe a full 3‐D distribution in 10 s. Thus, for many FTEs only a very few complete HPCA distributions are available. To provide greater sampling within the magnetosheath FTE, the longest intervals have been preferentially selected. It is noted, however, that this selection criterion introduces the potential bias that minor ion composition of only the slowest moving and/or largest FTEs are examined. Nevertheless, the availability of complete HPCA distributions at such temporal resolution is a distinct enhancement over what was previously possible from other missions  ≈ 257°)), which is conducive to the occurrence of reconnection and FTE production at the dayside magnetopause . The shear angle values are determined from the magnetic fields on the magnetosheath side  related to the reconnection process.The white arrow originating from the MMS location in Figure Bx, with a coincident enhancement (of a factor of ~2) in the field intensity. The time of peak magnetic field intensity (approximately 12:49:45 UT) marks the time of closest encounter with the center of the FTE. The total ion (ni) and electron (ne) number densities as measured by the FPI instrument on board MMS1 are shown in Figure Figure Figures + and O+ particles. In these two panels, the energy spectrograms displayed are the combined spectrograms as observed by the HPCA instruments from the four closely spaced MMS spacecraft. This is done to increase the low counting statistics of these species, to better understand the behavior of these ions. Prior to the first encounter with the core of the FTE at 12:49:24 UT, there were no energetic He+ ions. A few energetic O+ ions of energy >10 keV were observed immediately prior to this encounter . Within the core of this event, energetic He+ and O+ ions were both observed. At the trailing edge, the He+ ions disappear coincident with the more energetic antiparallel electron fluxes, while O+ ions continued to be observed at higher energies until such time that the more energetic parallel electron fluxes disappeared. The appearance of higher‐energy He+ and O+ at the edges  of the core of the FTE relative to the center of the core region is directly related to the gyroradius of the ions. Higher‐energy particles have larger gyroradii, and O+ at the same energy has a larger gyroradius than He+; explaining the appearance of O+ outside of the core region and further from the FTE core region than the He+ ions.Figures It is noted here that the proton count rate has been reduced by a factor of two in the MMS1 and MMS2 HPCA instruments by the RF attenuation setting within the instrument, while MMS3 and MMS4 proton reduction by the RF attenuation is even more stringent (a factor of 10). This is done to suppress the large number of protons  in order to better discriminate and observe the sparse minor ions. Nevertheless, some proton contamination still appears in the time‐of‐flight channels of heavier species at the lower energies.+ and O+ ions within the core of the FTE suggest that these field lines had undergone reconnection at an earlier time; though the presence of bidirectional energetic electrons indicate that the field lines of the core at the time of observation were closed. However, the continued presence of higher‐energy parallel electron flux after the higher‐energy antiparallel electron flux ceased to be observed indicate that reconnection to the southern ionosphere was still active at the trailing edge of the FTE. The minor ion densities and distribution functions during this FTE are next examined.To summarize the observations of Figure + and He++ number densities determined from each of the HPCA instruments from all four MMS spacecraft , the O+ density was slightly larger near the trailing edge of the FTE. The differences in these smaller number densities between the different instruments are due to count statistics, and because the half‐spin start times for each HPCA instrument are not coincident.Figures +, He++, He+, and O+). The time labels denote the start time of the 10‐s interval. The complete set of distribution functions over this and the subsequent FTE burst mode intervals for all species and all four HPCA instruments are provided in the supporting information.To further explore the characteristics of the populations of protons and the minor ion species to better understand the topological history of this event, sample 2‐D and 1‐D cuts of the full 3‐D particle flux distribution functions as measured by HPCA on board MMS1 are shown in Figure Bx > 0). The proton and alpha distributions are different because the shocked solar wind alpha population forms a shell distribution , when higher‐energy bidirectional electrons were observed. At this time the proton flux is strongly peaked parallel to the magnetic field, with a smaller peak at lower energy traveling antiparallel to B. The alpha particles are mixed and much reduced; much of the flux appears to be perpendicular to the magnetic field. The He+ and O+ distributions both show significant number fluxes traveling parallel to the magnetic field. Thus, the proton, He+, and O+ populations all traveling parallel to the field at approximately the same velocity are all likely of ionospheric origin. This suggests that the FTE core was topologically connected to the magnetosphere and, based on the schematic of Figure The top two rows display the particle distributions along the open magnetosheath draped field region (12:48:50–12:49:00 UT), prior to the FTE core encounter. It is seen that both the protons and alpha particle fluxes peaked along the direction antiparallel to B, as would be expected for solar wind flow from the Sun since the event occurred within a “toward” sector .The last two rows show the particle distributions during the brief interval when higher‐energy unidirectional electrons were observed moving parallel to the magnetic field (12:50:40–12:50:50 UT). In contrast to the electrons, the proton distribution was very similar to that of the draped magnetosheath population described in the first two rows . The alpha particle flux was more significant than in the core of the FTE but showed no clear peak relative to the magnetic field. HeFigure J × B and The formed flux rope would convect southward due to the magnetosheath flow and influenced by the 3.2Bx and negative By GSM components  were very similar to those of the previous FTE discussed  constructed from HPCA observations during specific, individual half‐spins of the MMS1 spacecraft. The first two rows show the distribution functions observed prior to the encounter with the FTE core (11:19:29–11:19:39 UT). Protons were observed with the peak flux slowly traveling antiparallel to the magnetic field. The very slow speed associated with the peak of the proton flux is consistent with the slow solar wind speed observed at Wind. Alpha particles were also observed with a peak flux that was ~1% of the proton peak flux, also moving antiparallel to the magnetic field. He+ and O+ ions were not observed at this time.Figure + and O+ ion fluxes were not significant.The next two rows show particle distribution cuts for a time interval near the FTE core as estimated from the peak magnetic field intensity (11:20:19–11:20:29 UT). The proton peak flux remained antiparallel to the magnetic field (in contrast to the first FTE case), but there was apparent preferential heating of protons moving parallel to the magnetic field. Alpha particles from the solar wind continued to appear only in the antiparallel direction, while the distribution cuts of He+ and O+ ion fluxes were again not significant.The final two rows of Figure + and O+ fluxes observed within this magnetosheath FTE from that observed in the first case may be due to whether a plasmaspheric plume  and duskward (By < 0), and the IMF clock angle is ~101°. The solar wind proton and alpha particle number densities , as was the solar wind bulk speed of 360 km/s  was southward and oriented mainly along a typical Parker spiral configuration as depicted in Figure s Figure  were rels Figure . The solThis isolated, long‐duration magnetosheath FTE was observed by the MMS spacecraft after apogee (similar to the previous two examples), again in the postnoon sector south of the GSM equatorial plane. Figure By. The location of MMS in this case (green circle) is therefore predicted to have been distant from the model reconnection line, and likely to have been distant from the FTE formation site as well. It is likely that this FTE had evolved and perhaps decayed significantly by the time it was encountered by MMS. The FPI ion bulk flow velocity (white arrow) observed near the center of the FTE core, drawn from the MMS location was nearly along the same direction as the ambient magnetosheath flow and consistent with an encounter with a reconnected flux tube that was preferentially connected to the southern ionosphere. The connection of the magnetosheath magnetic field with the magnetospheric field at MMS is crudely represented by the purple arrow and arc. In contrast to the first two cases, in this case the magnetosheath portion of the reconnected magnetic field vector points away from the noon meridian and points instead down the flank toward the magnetotail.Figure Figure The energy flux spectrograms of omnidirectional proton and alpha particle flux as observed by the HPCA instrument on MMS1 are displayed in Figures + and O+ from the HPCA instruments on board all four MMS spacecraft. Similar to the first FTE case (23 October 2015), the higher‐energy He+ ion flux shows a distinct energy dispersion wherein the highest energies appeared further from the center of the interval, and this is attributed to the ion gyroradius. The higher‐energy O+ ion flux showed a similar temporal variation but was less distinct. Observed counts at lower energies (less than ~1 keV) are proton contamination.Figures + and O+ ion partial densities (for E > 1 keV) are shown across the sampled FTE in Figures + density was a factor of ~10 smaller than the He+ density. In contrast to the earlier two cases, each of these magnetospheric ion species exhibit two distinct peaks at the edges of the FTE, with clear density depletions nearest the FTE center. It is noted that these peaks at the FTE edges were observed by all four HPCA instruments.Figure +, He++, He+, and O+}. The first two rows show the ion distributions cuts in the magnetosheath (15:45:54–15:46:04 UT), prior to the encounter with the FTE core. For this case, the flux peaks from all four species were observed moving along the direction parallel to the magnetic field. Protons and alpha particles were clearly observed, with peak fluxes traveling parallel to the magnetic field. Although this is counter to the previous two cases, it is consistent with the bulk flow of plasma away from the Sun. Very narrow flux peaks of He+ and O+ ions are also noted, but at these low energies are likely just proton contamination.Two‐dimensional and 1‐D cuts of the full 3‐D ion distributions from the HPCA instrument on board MMS1 are illustrated in Figure + and O+ ions were also both observed, also moving parallel to the magnetic field. These latter two species originated within the magnetosphere along field lines attached to the southern ionosphere.The next two rows show particle distribution cuts from the time of the first density peak of minor ions within the FTE core (15:47:14–15:47:24 UT). The proton distribution shows some heating of the protons parallel to the magnetic field; and a minor component of the flux was moving antiparallel to the magnetic field. Alpha particles were observed moving parallel to the magnetic field. HeThe last two rows show particle distribution cuts from the time of the second density peak of minor ions within the FTE core (15:48:14–15:48:24 UT). The distribution cuts are very similar to the previous time interval; although there is an indication of an additional small flux peak parallel to the magnetic field in the alpha particle distribution cut.+ and O+ moving parallel to the magnetic field are consistent with an FTE, which was magnetically connected to the southern ionosphere. In this respect, this reconnection scenario is similar to the first two FTE cases.The ion distributions of He3.4By component and a negligible Bx component . The solar wind speed was ~480 km/s  dayside kinetic challenge magnetopause crossing interval of 18 November 2015 as observed by MMS and previously analyzed by Kitamura et al. (E), based on HPCA ion distributions . A more comprehensive description of the relative distance between MMS and the magnetopause reconnection site during the GEM interval is provided in the companion paper of this special issue by Trattner et al.  were observed during the time interval of enhanced higher‐energy proton flux  and were both similar to the peak number density of higher‐energy O+ observed in the previous FTE case (20 October 2015).Figures  Figures  and 16f.+, He++, He+, and O+}. The first two rows show the ion distributions cuts near the observed start of the region of enhanced higher‐energy protons and electrons moving parallel to the magnetic field (12:48:19–12:48:29 UT). The distribution function of protons at this time was complex. A strong proton peak at a speed commensurate with the shocked solar wind flow (~100–200 km/s) was observed parallel to the magnetic field; similar to the previous FTE case. Another strong proton peak at ~700 km/s parallel to the magnetic field and represents protons reflected from the magnetopause at twice the local Alfven speed of ~210 km/s above the incident proton speed  is also noted. This is consistent with magnetospheric ions exiting the magnetosphere and imply a connection of the local magnetic field to the southern ionosphere.Two‐dimensional and 1‐D cuts of the full 3‐D ion distributions from the HPCA instrument on board MMS1 are illustrated in Figure + and O+ fluxes were significant at this time, with a single peak parallel to the magnetic field at ~400–500 km/s.The next two rows of Figure + or O+ flux was observed at this time.The last two rows show the constituent distribution cuts shortly after the trailing edge of the FTE, in the magnetosheath (12:50:19–12:50:29 UT). The proton distribution at this time was very similar to the magnetosheath distribution in Figure + and O+ densities indicates that this FTE has evolved from the time of initiation to the point of observation. While the larger‐scale behavior of protons and alpha particles show a depletion within the FTE relative to the ambient magnetosheath, a localized enhancement of these populations exists within the FTE—but not at the location of maximum magnetic field intensity. The localized density enhancement suggests that this event is the result of a coalescence of two FTEs.As with the previous intervals, the magnetospheric ion flows parallel to the magnetic field associated with this FTE are consistent with a magnetic connection to the southern ionosphere. However, the large distance of the observed interval from the model reconnection line and the relatively small He4Bx|. The solar wind ion densities and solar wind bulk flow speeds were in the range 4–12 cm−3 and 290–480 km/s, respectively. The MMS spacecraft were situated in very similar locations during each encounter, that is, within the magnetosheath close to the magnetopause, in the midafternoon local time sector and southward of the GSM equator. Our main findings are as follows:The presence of ions of magnetospheric or solar wind origin within the FTEs has been discussed. From this their magnetic connectivity to the IMF and the ionosphere has been deduced.+ and O+) flowing parallel to the magnetic field within the “core” of each FTE.The FTE observations have been related to dayside reconnection and reconnection line locations along the magnetopause according to the maximum magnetic shear model. All four events were predicted to be southward of the reconnection line and hence to have some magnetic connection to the southern ionosphere. This prediction was supported by the observation of magnetospheric ions , while the presence of a mixture of plasmas of both solar wind and ionospheric origin indicated that it had undergone reconnection in the past.The simultaneous presence of alpha particles, and HeFinite gyroradius effects in the time profiles of ions of ionospheric origin have been noted. This appeared in the form of energy dispersions. Thus, the most energetic ions, that is, those with the largest gyroradii, were typically observed in the outer regions of the FTEs.The variability of the minor ion content has been highlighted. Despite the similarities in solar wind conditions and the location of MMS, each of the four examined FTEs exhibited some unique features. The observed differences between these events demonstrate the challenges of understanding the occurrence, morphologies, topologies, and the evolution of FTEs. Table The ion composition in context with electron fluxes and magnetic field signatures of four long‐duration magnetosheath FTEs by the MMS mission occurring under similar steady or slowly varying solar wind conditions have been examined. All four events occurred during southward IMF. Three of the four cases were sampled during nominal Parker spiral conditions; while the fourth case was sampled during a time minimal IMF |To conclude, although flux transfer events have often been found to exhibit complex behavior, ion composition measurements examined in conjunction with electron and magnetic field observations help to better understand interactions between the solar wind and magnetosphere in these transient phenomena. Specifically, the minor ions help determine connection to the solar wind, magnetosphere, or both. The minor ions also help establish connections to ionospheric footpoint(s) (northern and/or southern polar region), useful for understanding the magnetic topology associated with FTEs. A larger survey of observed minor ion content associated with FTEs is expected to provide a more comprehensive understanding of how such transient events topologically connect the plasmas of the shocked solar wind and the magnetosphere.Supporting Information S1Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.


Explosively welded layered materials made of (a) an AA2519 aluminum alloy (AlCuMgMn + ZrSc), (b) titanium alloy Ti6Al4V and (c) an intermediate layer composed of a thin aluminum alloyed AA1050 layer are considered herein. This study presents test results connected to measurement science including microstructural observations of the material combined with the explosive method, and a basic analysis of the strength properties based on microhardness and tensile tests. Owing to the joint’s special manufacturing conditions, the laminate was subjected to deformation measurements with the digital image correlation (DIC) method. The research was supplemented by the residual stress measurements with the sin2ψ X-ray method based on the diffraction–reflection analysis that was verified by the bore trepanation method. In this article, a layered composite was tested which will be used as a construction material with increased ballistic resistance for spacecraft and military vehicles. In space technology, the 2519 alloy is used for protective panels against micrometeorites and for the construction of space station chambers , U0 is the input voltage [V], N is the bridge layout factor for the quarter bridge N = 1, K is the strain gauge constant and A is the output signal amplification.The resulting output signal registered in the respective depths of the bore was then converted to strain values in accordance with Equation (2).maxσ and minσ are the principal stresses, ε1, ε2 and ε3 are the strains measured on strain gauges 1, 2 and 3 and A and B are the coefficients that depend on the material properties, measuring rosettes and hole geometry, while α is the angle between strain gauge 1 and the direction of the nearest residual stress.As a result of the measurements and calculations, the characteristics of the relative deformation changes as a function of the depth of the hole were obtained. To determine the values of the main stresses and their direction, the following dependencies were used:The results obtained for selected measuring points are illustrated in the form of strain change courses as a function of the hole depth. minσ determined at all the measurement points in the case of the AA2519 alloy differ from each other (in the range of −121 to −104 MPa), while the maximum values of maxσ differences are in the range of −88 to −31 MPa. The observed differences in the values of minσ and maxσ are associated with stress value changes as a function of distance from the material surface. The orientation analysis of the principal stress vector showed that the direction of the minimum stress minσ was almost perpendicular with respect to the longer edge of the plate, while the maximum stress direction maxσ was parallel to this edge. The residual stress level in the aluminum alloy was very close to a homogeneous distribution (invariant as a function of depth). No tensile stresses were recorded. The stress values determined at all the measured points in the Ti6Al4V alloy were in the following ranges: minσ = −349–−184 MPa and maxσ = −111–−140 MPa. The obtained test results are associated with changes in stress values as a function of the measurement depth. The analysis of the residual stress orientation vector showed that the direction of the external load that affected the tested laminate was perpendicular to the longer edge of the plate. This caused higher compressive stresses that were similar to those observed during the measurements of the internal stress in the aluminum alloy AA2519.The minimum values of principal residual stress 3Ti precipitates were formed. This interlayer significantly changed the functional properties of the material. It was used to form ballistic panels in spaceships. Recognition of the stress values will allow for improving the numerical design in the phenomena of ballistic resistance [Explosive bonding enables the combination of hard bond materials, such as titanium and aluminum alloys. The combination of the proposed method generated an interlayer between the base materials in the microstructure. As a result of the short-term effects of the high pressure in the layer between the Ti6Al4V and AA1050 alloys, Alsistance . In addiIt should be noted that the stress values obtained in the titanium alloy may be subject to increased uncertainty. This is owing to the fact that titanium cannot be easily cut. Titanium tends to cause increased temperatures in the vicinity of the hole surface owing to the increased friction between the drill and the low-thermal conductivity titanium surface. The described difficulties caused an increase in the cutting resistance and interfered with the recorded output signal that manifested in the form of changes in voltage pulses toward negative values, i.e., caused tensile stresses. These assumptions confirmed that there was a significant increase in the cutting resistance that caused either a decrease in the rotational speed of the tool, or stopped the drill completely, followed by an abrupt increase in the output voltage. It should be stated that certain stress values in the titanium alloy may be altered, however, the degree of this disturbance is impossible to determine.


Gold/silver bimetallic nanoparticles still attract extensive interest due to their favorable properties e.g. in plasmonics or catalysis. We present here a facile and robust way for the production of clean Au/Ag binary nanoparticles (BNPs) with a total control over the composition via the spark discharge nanoparticle generation technique. With the application of pure Ag and Au electrodes, a tuning range of 55 to 90% Au content was achieved, but this can be further extended to the full 0 to 100% range by using a couple of alloyed electrodes. An added benefit of the approach is that either the concentration or the mean particle size can be kept constant at every composition by adjusting the generator parameters. Based on the systematic experimental data collected, a semi-empirical model for the prediction of the Au/Ag BNP composition was also developed. This model was used to calculate the theoretically achievable Au/Ag composition at a given spark parameter set in the parameter range most commonly used in the literature. Due to their broad field of applications covering e.g. photothermal therapy2, localized surface plasmon resonance (LSPR) based sensing5, or surface enhanced Raman spectroscopy8, tremendous work has been done to control their properties and hence their performance. Gold and silver NPs have been synthesized with various shapes including spheres, rods, cubes, boxes, triangles or stars with sizes ranging from a few nanometers to tens of nanometers11. Mixing these two metals opens up further perspectives in their LSPR-related applications. Au/Ag alloy NPs have a plasmon resonance band between the plasmon bands of pure gold and silver, the position of which can be continuously varied by controlling the alloy composition12. In addition to this tunability, Au/Ag alloys effectively combine the high activity of silver with the excellent chemical stability of gold13. Au/Ag alloy NPs have found successful applications in e.g. the field of surface enhanced Raman spectroscopy14, biomedical imaging12 or catalysis16.Gold and silver nanoparticles (NPs) serve as the workhorses of plasmonic research for decades due to their strong plasmon resonance, which can be excited by visible light17. In order to have more control over the reproducibility and size distribution of the NPs, seed-mediated synthesis is often used where the nucleation and growth stages of particle formation are separated12. Additional ways to produce Au/Ag alloy NPs via chemical synthesis can be carried out e.g. via microwave heating in a polymer solution18, ultrasonic treatment19, or photochemical synthesis20. Physical methods offer relatively simple alternatives to chemical synthesis, while avoiding the use of reagents, solvents or various precursors. These are typically based on the ablation of a bulk alloy via a laser21 or spark discharge22. However, the preparation of a bulk target with a predefined composition is often impractical when the tuning of the particle composition is desired.The literature describes many possibilities for the synthesis of Au/Ag alloy NPs via either chemical or physical methods. Among all the synthesis routes, co-reduction of Au and Ag precursors is probably the most popular and straightforwardIn the present paper we report on the gas phase synthesis of Au/Ag alloy NPs in various compositions via spark discharge generation, an inherently clean and environmentally friendly method. We demonstrate and investigate the potential of spark discharge nanoparticle generators (SDGs) for the continuous tuning of the composition of Au/Ag alloy NPs, by simply changing the resistance of the discharge circuit. The achievable composition range as well as the underlying physical processes are discussed. The compiled experimental data and theoretical considerations are used to construct a semiempirical mixing model, which can be used to predict the average composition of the Au/Ag BNPs generated in an SDG.23. As a result of the accompanying high current, the electrode material is released into the gas phase where, after nucleation and growth, NPs are formed24. If the electrodes are dissimilar, particles with complex composition can be obtained. The particles formed in the process are proven to contain material from both electrodes, as a consequence of the erosion of both electrodes26, which stems in the oscillatory nature of spark discharges27. The current waveform of a spark follows an exponentially dampening sinusoidal function. This corresponds to the fact that the discharge circuit of an SDG is essentially a serial RLC circuit, where the current is described by the well-known Eq.  changes in every half cycle. Since the material erosion caused by a spark discharge is mainly attributed to the arc stage of the discharge28, which is the high-current conducting phase of the spark, polarity reversals result in an alternating erosion of the electrodes. As a result, atoms from both electrodes will be released into the gas phase and will serve as the source for the formation of nanoparticles. It is well known that different electrode materials have different erosion rates during sparking30; this will also affect the composition of the generated BNPs. Typical morphology of the particles formed in our generator is shown in Fig. 22. Please note that the other peaks in the EDX spectrum are originating from the TEM grid used for sampling and the vacuum chamber. Samples shown in Fig. 30. It should be noted that the composition of the SDG-generated BNPs exhibit a certain distribution, meaning that the composition of an individual particle may differ from the average composition, which is originated from the peculiarities of the spark plasma-based process31.SDGs are based on the initiation of an energetic spark discharge between two electrodes, which are placed in a controlled, flowing—usually inert—gasnown Eq. .1\docume30, which can be taken as the manifestation of that material removal mainly takes place at the cathode. This in turn means that the relative erosion rate of different electrodes will also depend on their initial polarity32, which leads to the conclusion that the composition of the generated BNPs can be altered by simply switching the polarity of the electrodes. This, however, only allows for a certain constant shift in the composition. For example, under experimental parameters corresponding to Fig. 32. It means that by altering the symmetry of the current waveform, the relative erosion of the electrodes and hence the composition of the BNPs can be tuned.It was mentioned in the previous subchapter that the composition of BNPs generated via sparking is affected by the erodibility of the two electrode materials. Nevertheless, this is not the only factor that influences the particle composition. In most cases, the erosion of the initially cathodic electrode is reportedly larger than that of the anodic electrode, even in case of identical chemical compositionThe current waveform can be altered by modifying the resistance, inductance, and/or capacitance of the discharge circuit. Experimentally, the most accessible parameter is resistance. By looking at Eq.  it is ap33. When the total resistance is increased, the peak current decreases and the dampening increases, resulting in a lower number of current reversals and a shorter duration of the sparking. It can also be seen that along with an increasing damping, the asymmetry also becomes more pronounced, which is related to the energy delivered to the electrodes in subsequent half-cycles, proportional to the current. For the description of “the degree of asymmetry” and hence the ratio of energy delivered to each electrode in their cathodic periods, Feng et al.31 introduced the k factor:Ipositive is the current measured in the half-cycles when it has a positive value and Inegative when the polarity is reversed, given that the current probe is installed in a way that the current is positive in the first half-period. As is shown in Fig. k factor increases along with the increase of the circuit resistance. This means that by changing the resistance of the circuit, a continuous tuning of the current waveform can be achieved.The effect of increasing circuit resistance on the current waveform is exemplified by Fig. k factor on the composition, Au/Ag nanoparticles were generated with the systematic increase of the circuit resistance, for both electrode polarities. Results are shown in Fig. k factor  electrodes is maintained by the generated BNPs during sparkingk factor. For the prediction of the composition of BNPs generated in SDGs, Feng et al. proposed the following semi-empirical formula31:CA and CC are calculated from thermal constants of the initially anodic and cathodic electrode, respectively31. The Feng model is based on two main assumptions: i) the eroded mass is linearly proportional to the energy delivered to each electrodes, described by the integral of the square of the current, and ii) only the electrode which is momentarily the cathode is eroded31. In Fig. k factor calculated for one of the two cases. Here we set k to be greater than 1 when Ag is initially cathodic, which brings it to between 0 and 1 when Au is initially cathodic. It can be seen that although the model provides a reasonable prediction for the trend as a function of the k factor, the calculated values are far off from the measured values. The relative deviation from the experimentally determined compositions is in the range of 10–65% and the model consistently underestimates the gold content. This could mean that either the CA and CC constants fail to properly describe the “erodibility” of the electrode materials, or the energy input is not describable solely by the integral of the square of the current for every pair of electrode materials. A physical explanation for the latter case can be given based on the so called sheath layers of a gas discharge plasma existing in the vicinity of the electrodes36. Energy exchange between the plasma and the electrodes is mostly governed by these regions which responsible for the voltage drop over the discharge39. The energy delivered to the electrodes can be calculated by integrating the product of the voltage drop and the current40. It should be noted that formally a resistance can be assigned to the sheet layer and hence the energy can be calculated from the integral of the square of the current as in the Feng model. Nevertheless, the voltage has much more physical relevance and therefore literature values can be usually found for the voltage drop. Literature data show that the exact values for voltage drop over a discharge gap depends on the electrode material and the ambient gas as well. Values in the range of about 14–50 V can be found for various metals, which are independent from the current over a relatively broad current range42. If we consider first that only the momentary cathode is eroded in an SDG—in accordance with the Feng model—the typical cathode fall voltages should be compared for Au and Ag in the present case. The most recent value we found for silver is about 14 V38, but unfortunately we are unaware of any relevant measurements in case of gold electrodes. Nonetheless, if we assume that cathode fall voltages are different for gold and silver, the different energy input and hence different erosion rates would explain the strong deviation of the calculated composition values from the measured ones  eroded can be also looked at. Our ICP-MS analytical data obtained for the average composition of the BNPs allows for a more accurate evaluation of the eroded masses than simple gravimetric measurements typically used in SDG research. Based on this, we are able to compare the mass of Au and Ag converted to aerosol (that is in the portion of aerosol collected on the filter) when either the Au or the Ag electrode is initially cathodic. Assuming that the current is reasonably reproducible at a given total resistance—which is a condition generally fulfilled—the particle mass ratio generated from the initially cathodic and anodic electrodes (denoted as mcathode/manode) can be obtained from the two measurement sets shown in Fig. mcathode/manode should be equal to k for a given electrode material, according to Eq. , inductance (L), and the capacitance (C) of the discharge circuit which can affect the current waveform and hence the k’ factor. These parameters jointly determine whether the spark operates in the underdamped—i.e. oscillatory—or overdamped—i.e. unipolar—regime, therefore setting practical limits to their values in an SDG. From a technical point of view, R and C are the most conveniently accessible parameters, hence here we only investigate their effect on the Au/Ag composition. We assume an inductance of 1 µH, which is characteristic to our setup and also close to the values reported in the literature33. Typically, SDGs are built with—sometimes variable—capacitance in the range of ca. 1–30 nF44, which determines that the transition between the oscillatory and unipolar regimes occurs at a maximum of about 11 Ω. As it was mentioned earlier, the lower limit of the total resistance is defined by the minimum resistance of the circuit and the spark plasma, which in total is usually not much less than 1 Ω. These values set a rational parameter range for an SDG. The theoretically achievable composition values calculated from Eqs.  with varying composition. The resistance of the discharge circuit was varied in order to change the relative erosion of the Au and Ag electrodes and therefore change the composition of the generated BNPs. The dependence of the average composition of the BNPs on the total resistance and initial electrode polarity was experimentally determined via ICP-MS analysis. It has been shown that the Au content can be continuously varied between 55 and 90 m/m%. By replacing one of the electrodes with an alloy of the two elements, practically the whole 0–100% composition range can be covered. This convenient composition tuning can be achieved without altering the particle size, as proved by the in situ size distribution measurements.In order to elucidate on the relation of the experimental parameters and the BNP composition, the experimentally acquired data was compared to an existing spark plasma mixing model. This comparison revealed considerable deviations between the measured and calculated values, which led us to reconsider some of the assumptions generally used in the explanation of spark mixing. As a result, a plausible physical explanation was given to the underlying processes, and an extended mixing model was created to predict the Au/Ag BNP composition. This means, that our experimental technique for producing Au/Ag BNPs is also backed up by a validated semiempirical model, which further supports the effective parameterization of experiments aiming to the generation of Au/Ag BNP of virtually any target composition via an environmentally friendly, facile, one-step gas-phase route.45 and schematically shown in Fig. The experimental setup was already described elsewhereThe argon  carrier gas flow was fed in the chamber via the down KF-40 port (upward pointing “crossflow” with injector nozzle), which was employed during NP generation. The gas flow rate was controlled by a mass flow controller  and set to 5 standard liter per minute (slm). All experiments were carried out at atmospheric pressure, monitored by a piezo-resistive pressure gauge .The spark discharge was created by a simple capacitor charging circuit. A monolithic, high voltage capacitor  with 8 nF capacitance was connected parallel to the spark gap and was charged by a high voltage power supply . The spark discharge between the electrodes is created when the voltage on the capacitor reaches the breakdown voltage of the gas between the electrodes. The resulting spark discharge is a bipolar, dampened, oscillatory discharge. The repetition rate of the spark discharge  was controlled via the charging current of the capacitor and kept constant at 100 Hz during the experiments, unless stated otherwise. In our measurements the total resistance of the electric circuit was varied by different length of FeCrAl alloy resistance wires  in the range of 1–9 Ω. The voltage and current waveforms in the discharge loop were recorded by a 200 MHz digital storage oscilloscope  using a broad-band high voltage probe  and current probe , at the same time.46. To this end, an inductively coupled plasma mass spectrometer  was used. Sample dissolution was carried out by aqua regia, prepared freshly from trace quality cc. hydrochloric and cc. nitric acids  under 16 h of contact time. The resulting clear solutions were filtered through 0.22 μm PTFE membrane filters and diluted with trace-quality de-ionized labwater  prior to analysis. Multipoint, matrix-matched calibration was performed using certified calibration standards . ICP-MS plasma and interface parameters were optimized via standard tuning solutions . All measurements were carried out by monitoring the signal of the 107Ag and 197Au isotopes, in He mode using the ORS3 collision cell. Data processing was performed within the Agilent Mass Hunter software. The 99.996% purity argon and 99.999% purity helium gases were purchased from Messer Hungarogáz Kft.The created alloy NPs were collected on a glass microfiber filter  in a filter holder . The sample collection time was 0.50 h and between every sample the plastic tubes and the filter holders were cleaned with a mixture of 50% ethanol  and 50% water solution in an ultrasonic bath . The resulting samples were stored in Petri dishes until composition measurements, which were carried out by following the same procedure we have already utilized for SDG-generated Au/Ag NPsFor TEM and EDX analysis, the generated particles were sampled using a lacey carbon copper grid  by using a low-pressure impactor. The morphology of the nanoparticles was analyzed by high-resolution transmission electron microscopy . The energy dispersive X-ray  profiles were obtained at an accelerating voltage of 200 kV.The size distribution of the spark generated particles was measured using a scanning mobility particle sizer (SMPS) system consisting of an electrostatic classifier , an ultrafine condensation particle counter , and an aerosol charge neutralizer . The SMPS system was operated with a sample flow of 0.3 L/min, a sheath flow of 3 L/min, and a scan time of 60 s (measurement range: 4.61–151.20 nm).Prior to size distribution measurement and particle sampling for morphology characterization the generated aerosol passed through a compaction furnace  equipped with a ceramic tube set to 900 °C in order to compact the produced nanoaggregates. It has been confirmed via ICP-MS analysis that the compaction did not change the average composition of the generated particles collected on a filter.Supplementary Information.


The presence of this glycosylated protein in serum is a fraction of glycogen that is degraded outside the lysosome and the cell. The glycosylated protein in the serum is not present in the serum of Pompe mice not on ERT, but it is present in the serum of Pompe disease patients who are on ERT, so it is a biomarker of GAA degradation of lysosomal glycogen.In studies on the degradation of glycogen by rhGAA, a glycosylated protein core material was found which consists of about 5–6% of the total starting glycogen. There was an additional 25% of the glycogen unaccounted for based on glucose released. After incubation of glycogen with rhGAA until no more glucose was released, no other carbohydrate was detected on HPAEC-PAD. Several oligosaccharides are then detectable if the medium is first boiled in 0.1 N HCl or incubated with trypsin. It is present in serum either in an HCl extract or in a trypsin digest. The characteristics of the in vivo serum material are identical to the material in the in vitro incubation medium. One oligosaccharide cannot be further degraded by rhGAA, from the incubation medium as well as from serum co-elute on HPAEC-PAD. Several masked oligosaccharides in serum contain  The deficiency of the lysosomal of α-glucosidase (GAA) in Pompe disease tissue, also known as type II glycogenosis and acid maltase deficiency, was identified in 1963  and its Enzyme replacement therapy using recombinant human GAA [rhGAA] for Pompe disease has been facilitated by the development of the knockout mouse model which is deficient in the lysosomal α-glucosidase . There aThe initial goal of this work was to determine if rhGAA could completely degrade glycogen. A core glycosylated protein, which is glycosylated primarily with inositol and sorbitol, iditol and has minor constituents of glucose, galactose, and mannose, as well as galactosamine and glucosamine, was identified. The mass of which consists of about 5–6% of the initial glycogen in the incubation tube . It is lSigma, Type IX bovine liver glycogen, SigmaAldrich, St. Louis, MO, USA, is extracted by the method of Bell and Young,  which inHuman glycogen samples were extracted by the method of Mordoh, Krisman, and Leloir  with theAutopsy liver tissue from an 18-month-old female with Pompe disease (type II glycogenosis) and liver tissue from two adult male accident victims. The Pompe liver and the Control 1 liver were obtained at autopsy. In the case of Control 2, the patient was an organ donor on life support so the liver tissue was obtained immediately on termination of life support. All liver tissue was stored at −76 °C until the glycogen isolation. The case of the Pompe disease patient and an enzyme replacement trial with lysosomal α-glucosidase linked to low density lipoprotein has been previously reported .The IRB approval was UC Irvine, UCI/ 2008-6631, and the genomic analysis of patients was reported .g for 5 min to precipitate any insoluble material. Then, a 100 µL or 200 µL aliquot was extracted and boiled for 5 min. The sample was then centrifuged at 16,000× g for 5 min to precipitate any insoluble material and the supernatant was analyzed for carbohydrates by HPAEC-PAD on a PA1 column. The remaining incubation mixture was mixed on a vortex mixer and returned to the water bath.Recombinant human GAA (rhGAA) was provided by Sanofi Genzyme, Framingham, MA, USA, which is the 110 kDa precursor which is converted to the mature form in the tissue in ERT. Assay mixtures consisted of 1 mL volume containing 500 µg or more of glycogen as indicated, 50 mM sodium acetate buffer, pH 4.6, and 10 µL or 25 µL of rhGAA (5 µg/µL) as indicated. The reactions were incubated at 37 °C under toluene to prevent microbial growth. At various time points, as indicated in the figures, the reaction mixture was mixed on a vortex mixer, then centrifuged at 16,000× HPAEC-PAD was performed on a Dionex DX-600 ion chromatograph using a CarboPac PA1 column.  The eluent was 150 mM sodium hydroxide, isocratic from 0 to 5 min, then a linear sodium acetate gradient from 5 to 25 min going from 0 to 57% 500 mM NaOAc in 150 mM NaOH at a flow rate of 1 mL/min. Fractions of 0.25 mL were collected using a Gilson 201 fraction collector. Fractions were partially reduced in volume on a Speed Vac to a volume less than 1.0 mL and then dialyzed overnight against 18.3 megohm water in 1.0 mL chambers against a 500 MWCO membrane. Fractions were taken to dryness in a Speed-Vac. The fractions were then hydrolyzed with 2 N TFA at 100 °C for two hours after which they were taken to dryness in a Speed-Vac. If it was determined that hydrolysis was incomplete, as evidenced by changes on passage through a Dowex column, samples were hydrolyzed again with 4 N TFA at 120 °C for 1 to 4 h. Monosaccharides and sugar alcohols were determined using a CarboPac MA1 column with isocratic elution with 480 mM NaOH at a flow rate of 0.4 mL/min. The waveform for carbohydrate analysis had a potential of +0.1 V from 0 to 0.40 s, −2.0 V from 0.41 to 0.42 s, +0.6 V from 0.43 to 0.44 s, and −0.1 V from 0.44 to 0.50 s with integration from 0.20 to 0.40 s. Data analysis was performed using Dionex Chromeleon 6.60 software.Protein determination was by a modification of the method of Lowry et al. . A contrDemonstration of the oligomers in glycogen and their relationship to degradation by rhGAA is shown in Reaction mixture from rhGAA degradation of Control 2 glycogen was subjected to the scheme shown in HPAEC-PAD does not reveal any significant peaks that elute after monosaccharides. Which indicates no carbohydrates with ionizable hydroxyl groups are present?Extraction with 0.1 N HCl at 100 °C for 30 min reveals maltooligosaccharides from DP 2 to about 18 on HPAEC-PAD.4OH. This is indicative of binding by a charged species such as protein or amino acids. After taken to dryness, it can be extracted with 0.1 N HCl at 100 °C to reveal the maltooligosaccharides.The material in the incubation medium binds to a Dowex 50W ion exchange column and elutes in 2.0 N NHIncubation with amyloglucosidase does not do anything to the samples.Incubation with trypsin reveals some smaller oligosaccharides that elute in the region of up to about DP 4 and one at about DP 7 or 8. Additionally, trypsin treatment before HCl extraction appears to facilitate the appearance of more larger oligosaccharides. This is indicative of oligosaccharides being released or their appearance facilitated by the removal of protein.Incubation with concanavalin A does not appear to bind the material. This indicates the absence of exposed glucose or mannose residues, including glycogen, which would be bound by the concanavalin A protein .Lack of chemical detection of ionizable hydroxyls of carbohydrate.Lack of biological recognition of carbohydrate by rhGAA, concanavalin A, or amyloglucosidase.Binding to Dowex 50W indicative of a charged species.Exposure of carbohydrate by incubation with a protease (trypsin).These characteristics led to the conclusion that the material contains carbohydrate material which is masked by protein. There are reports in the literature of carbohydrate masked by protein. Since the material was not detected to be carbohydrate chemically, or by glycosidases and concanavalin A, it is possible that it is not recognized by the biological system. It was considered to be possible that it could be released outside the cell by the lysosomal exocytosis mechanism in which the lysosomal membrane fuses with the cell membrane and the lysosomal contents are expelled from the cell ,25. Thisg and allowed to clot. The serum was then diluted 1:10 and 1:20 and analyzed by HPAEC-PAD directly as well as after extraction with 0.1 N HCl for 30 min at 100 °C. The serum, HCl extract, and HCl extract following in vitro incubation of glycogen with rhGAA are shown in About 200 µL of blood was obtained from a fingertip needle stick of a normal individual and added to 300 µL of 0.9% NaCl in a conical 1.5 mL tube and immediately centrifuged for 10 min at 10,000× The HCl extract of the clot shown in From the initial observation of the in vitro degradation of glycogen, that the apparent absence of oligosaccharides in the incubation medium could be overcome by boiling in 0.1 N HCl for 30 min or by trypsin, the concern became one of the comparison of the methods. An overnight or 24 h incubation with trypsin did not reveal as much of the terminal oligosaccharide, which is not degraded by GAA as was released by the HCl treatment. However, longer trypsin incubation releases much more of the material as shown in The effect of doubling the concentration of trypsin used as well as chymotrypsin was tried. In 48 h incubation, doubling the trypsin concentration did not have a noticeable effect on the result. Chymotrypsin was more effective at the same concentration as trypsin. Proteinase K was also tried but there is a problem with proteinase K since it contains a number of oligosaccharides in the enzyme preparation which makes it problematic for collection of fractions.m-inositol, e-inositol, sorbitol, and xylitol (tentative) as well as hexoses and less xylose in 2-2 as shown in the bottom chromatogram for each fraction. During acid hydrolysis to release carbohydrates, although some carbohydrates are released with increasing time, others may be degraded. In this case, during the 4 N TFA hydrolysis, some glucose and a significant amount of xylose are degraded with increased time. This final TFA hydrolyzate is shown for both fractions in the bottom panel of Six fractions were collected from a trypsin incubation mixture which are labeled 1–6 in the top panel of It is apparent from this sequence that the initial fractions from the collection in the 150 mM NaOH/NaOAc elution medium are altered by the dialysis to remove the salt. Those peaks are then not apparent when the dialyzed sample is chromatographed. However, TFA hydrolysis of the fractions demonstrates that the material was present but that it was masked. Therefore, it appears that the initial in vivo material from serum or the incubation medium from in vitro rhGAA degradation is masked by protein. After proteolysis with enzymes, there apparently is still enough peptide material attached to mask the carbohydrate after dialysis. It may be possible to remove more peptide material by using proteases with different specificities. It appears that after initial hydrolysis with 0.1 N HCl at 100 °C for 30 min to expose the carbohydrate, the removal of salt by dialysis then permits a configuration change to again mask the carbohydrate. This is the case for both the material from the in vitro rhGAA incubations and the in vivo material isolated from serum.The three monosaccharide chromatograms for Fraction 1(2-1) after hydrolysis in 2 N TFA, followed by passage through a Dowex 50W column and hydrolysis in 4 N TFA are shown in The three monosaccharide chromatograms for Fraction 2(2-2) after hydrolysis in 2 N TFA, followed by passage through a Dowex 50W column and hydrolysis in 4 N TFA are shown in From the carbohydrate composition of the fractions it appears that the major components are the two inositols, sorbitol, xylitol, and mannitol are relatively similar and that the variability occurs in the monosaccharides. However, it is important to keep in mind that these are the monosaccharide compositions of oligosaccharides that still have some peptide attached. On incubation with rhGAA, all of them except 2-2 are degraded with an increase in 2-2 and free glucose, which indicates that although glucose is only a minor constituent, it likely is in a critical position. This is suggestive that at least a portion of the other glycopeptides are being converted to 2-2. There is still peptide attached but it is not known if the peptide is the same for all of them so it is not yet possible to determine with absolute specificity the quantitative interrelationships. It is very likely that there are multiple glycosylation sites, each having a different monosaccharide composition as will be discussed later.The question of whether these in vivo fractions are intact components of glycogen or whether they have undergone some modification by GAA, or any other enzymes, is an open question since GAA does have glucanase, glucantransferase, and glucosyltransferase activities under the same conditions in which it has glucosyl hydrolase activity ,6,7,8,9.The carbohydrate composition of these soluble glycosylated proteins is unique by consisting mainly of inositols and sorbitol with some iditol. Inositol and sorbitol are not known to be found on any other protein. Literature searches do not reveal any glycosylated proteins published with these as the major carbohydrate. In fact, a search does not reveal any publication of a glycosylated protein with sorbitolA summary of the various fractions isolated following in vitro degradation of glycogen by rhGAA as well as the fractions isolated from normal serum, in vivo, is shown in A soluble fraction of glycogen that is not completely degraded in vitro by rhGAA contains at least 20% of the initial glycogen. The fact that the carbohydrate cannot be detected unless the incubation medium is boiled in 0.1 N HCl for 30 min or incubated with a protease such as trypsin or chymotrypsin for up to 48 h indicates that the carbohydrate is masked by protein. If the carbohydrate was exposed on the surface of the protein, it would be detected. Following protease incubation, all but one of the oligosaccharides can be degraded or modified in vitro by rhGAA. The one major oligosaccharide in serum that cannot be degraded in vitro by rhGAA following the HCl or protease treatment persists as a terminal metabolite of in vivo GAA degradation of glycogen which is peak 2-2 in Perhaps if calibrated with the proportion of glycogen degraded, this terminal metabolite could possibly be useful to quantitatively assess lysosomal glycogen degradation in vivo. However, since glycogen is not homogeneous, such a determination may be difficult to achieve without a better understanding of the structure of glycogen. It should be kept in mind that such experiments would not account for ongoing replenishment of lysosomal glycogen by phagocytosis which is assumed to be the case for turnover in lysosomal function in tissues and has been demonstrated in fibroblast culture . One wouIt will be important to investigate the site of degradation of this glycosylated protein as it may be useful in the study of normal lysosomal glycogen in muscle. Liver or kidneys are likely sites of degradation. There is a neutral α-glucosidase in liver which is known to degrade maltooligosaccharides but it can not degrade glycogen . The endGlycogenin is a self glucosylating protein which acquires 10–20 glucose residues, which then serve as the primer for the glucosyl residues of glycogen . The glyComparison of the carbohydrate portions of unknowns 2-1 to 2-6 indicate that all have protein, inositols, sorbitol, mannose, glucose, and galactose and three appear to have xylose. The identification of xylitol is tentative.During this work, the reasoning was that if the carbohydrate could not be detected by either chemical or biological means, then perhaps the cell could not recognize it as carbohydrate either. As mentioned earlier, there are precedents for the lysosomal exocytosis following degradation of a substance up to a point at which the lysosomal membrane fuses with the cell membrane and expels the material from the cell ,26,27,28These results contribute to the observations of degradation of lysosomal glycogen. The question of where this soluble glycosylated protein is ultimately degraded is added to the list of unknowns as well as the site of degradation of the masked maltooligosaccharides. The fact that this soluble glycosylated protein has protein masking carbohydrate is significant. Such masking is known in biochemistry but I have not found any report for glycogen or for serum components ,14. To wIt is important to understand the masking mechanism to be able to quantify the soluble glycosylated protein as well as to understand the role of lysosomal glycogen in normal metabolism. Does this masking affect our methods of glycogen determination using amyloglucosidase? Should a proteolytic step be used before the amyloglucosidase degradation? These are now questions to be considered and experiments to be performed.It is likely that the glycosylated protein residue from degradation of glycogen by rhGAA in vitro, the glycosylated protein associated with the masked oligosaccharides, as well as the glycosylated protein terminal glycogen degradation product are all the same protein based on their carbohydrate composition and possible location of their association with glycogen as indicated in One of the most interesting points of these results is that the glucose and sorbitol content of these polymers is greater than the quantity of free glucose in the same volume of blood. It is likely that one reason this has not been reported is due to the masking. There also appears to be some self-assembly of protein/carbohydrate when isolated fractions are dialyzed which also complicates things. This self-assembly is not unknown in polysaccharides. A TFA hydrolyzate of serum indicates that one of the most abundant carbohydrates is sorbitol, which is more abundant than glucose.m-inositol was liberated by the passage of the sample over the Dowex 50W column of the 2 N TFA hydrolyzate and then much more by hydrolysis with 4 N TFA of the material passed over the Dowex 50W column. This is also true of myo-inositol in glycosylphosphotidyl inositol [GPI] anchor proteins. One reason that inositol and sorbitol may not have been reported as components of polysaccharides such as cellulose, starch, and glycogen may be that much of the earlier work was done using GLC where, for volatility, monosaccharides were converted to alditol acetates and m-inositol was added as an internal standard [A similar glycosylated protein has been found in cellulose and corn starch, which also contains mannose, glucose, and galactose, with the major components being inositols, iditol, and sorbitol . Sorbitostandard .This terminal metabolite from the degradation of glycogen by GAA and its masking by protein will provide a mechanism as a biomarker by which to gain direct information on the function of lysosomal glycogen and perhaps its role in muscle physiology. Up to this point, our understanding of lysosomal glycogen has been limited to its accumulation in Pompe disease. Perhaps now we can begin to understand the role of lysosomal glycogen and if the impairment if its degradation pathway contributes to the pathophysiology of Pompe disease in a manner different than that attributed to the accumulation of glycogen. This information will permit a better basic understanding of Pompe disease and a better understanding of ERT and how it mimics the normal lysosomal glycogen pathway as well as how it may differ from that pathway if, in fact, it does differ.This biomarker will enable an assessment of the effectiveness of ERT in Pompe patients in a relatively short time period of days. It could be monitored easily since only a pinprick is required for the small amount of blood needed. It will be possible to determine the most effective frequency of enzyme administration, particularly if it is found to vary among patients. The current two week interval may not be optimal for all patients so this will enable an assessment of GAA activity during the interval between administrations to assess the enzyme activity over the time period.The breakdown of glycogen by rhGAA in vitro only releases enough glucose to account for about 75% of the mass of starting glycogen. About 5–6% is attributed to a glycosylated protein core which has inositols, sorbitol as the major carbohydrates, as well as glucosamine, galactosamine, glucose, and galactose. About another 18% is released as maltooligosaccharides masked by protein and the remainder is a soluble glycosylated protein also masked. The masked carbohydrate and the soluble glycosylated protein are found in normal serum, which is evidence that degradation of about 25% of the lysosomal glycogen occurs outside of the lysosome and outside of the cell. This appears to be facilitated by lysosomal exocytosis. The soluble glycosylated protein is a terminal degradation product of GAA degradation of lysosomal glycogen. This soluble glycosylated protein is not present in the serum of Pompe mice which are not on ERT but it is present in the serum of Pompe patients on ERT and normal individuals. This soluble glycosylated protein is a biomarker for degradation of glycogen by GAA and should be useful to assess ERT in patients. Since the glycosylated protein appears to be a terminal degradation product of GAA degradation of glycogen, it has potential to be very useful to monitor such degradation in relatively short time periods. As such, it should be possible to monitor the degradation of lysosomal glycogen essentially on a daily basis, if desired, to monitor such activity in the interval between administrations of rhGAA. This should enable a better understanding of the whole ERT process and may lead to improved treatment practices.