This study explored the effect of substituting wheat flour (WF) with distinctive optimal doses of buckwheat flour (BF) corresponding to large, medium, and small particle sizes (PS), previously established based on an optimization process, on composite flour characteristics, dough rheology, and bread quality. The optimal dose for each PS was established in a previous study. The highest value for protein, lipid, mineral, and amino acids was found in the optimal composite flour with medium PS, with significant differences between those with large and small PS. The addition of BF in WF at doses corresponding to each fraction provides optimum rheological properties, with the large and medium PS providing higher performance compared to the small one. The same tendency was observed for volume and texture parameters of bread made from optimal composite flours with medium and large PS, respectively, but the crust and crumb lightness presented lower values than bread with small PS. Regarding the bread nutritional profile, the sample with medium PS possessed the highest protein, lipid, and ash content. Compared to the wheat bread, a considerably higher amino acid content, up to 21.22%, was found in bread made from optimal composite flours with medium and small PS, respectively. The bread samples with medium and large PS, respectively were superior in minerals, the value being up to 2.63 times higher compared to the control. Sensory characteristics results revealed that the bread samples containing 9.13% large and 10.57% medium PS were the most preferred by panelists. The results of this research make an important base to suitably develop wheat–buckwheat bread applications in the future. In general, bakery products made with refined wheat flour present a deficit of essential amino acids and minerals and must be supplemented to acquire daily physiological demands ,2. SupplPrevious research revealed that the incorporation of whole pseudocereal flour, especially in large amounts, decreased yield, volume, fermentation parameters, crumb elasticity, and modified the flavor, depending on pseudocereals and type of bread, because of the gluten matrix dilution ,12,13. MThe partial replacement of wheat flour with buckwheat flour in bread has been studied by different groups of researchers ,16,17,18A bread sensory properties evaluation demonstrated that buckwheat flour incorporation in wheat flour can potentiate the quality attributes of overall acceptability, specifically taste, color, and smell .2 production by baker’s yeast during fermentation [The influence of yeast in the preparation of buckwheat bread was researched by Moroni et al. ,25. Exteentation . Howeverentation . Gluten entation . Pseudocentation ,28,29. Bentation  affirmedentation  stated tentation .m), total volume of gas produced (TV), volume of the gas retained in the dough (VR), and retention coefficient (CR); rheometer parameters—storage modulus (G′), viscous modulus (G″), loss tangent (tg δ), maximum gelatinization temperature (Tmax), maximum creep-recovery compliance ; as well as bread volume (BV), and bread hardness (BH). Additionally, the optimal wheat–buckwheat composite flour coresponding to each fraction was established by applying multiple response analysis to the predictive models generated for empirical and dynamic rheological properties of the dough, as well as on some of the technical characteristics of the bread, in conjunction with the desirability function approach. For this purpose, by imposing the following desired goals for responses: ST, C3-4, H′m, TV, VR, CR, W, and Jrmax set at maximum value; minimization of C1-2, C5-4, P/L; and the level of all remaining responses maintained within range, the maximum dose possibility for each fraction which can substitute wheat flour and give the best technological characteristics for composite flour and bread quality was established.In our previous study , we inveThis paper continues our previous work and presents a complete characterization of the optimal products obtained in there. To our knowledge, there is a scarcity of research about the influence of the optimal wheat–buckwheat composite flour containing large, medium, and small buckwheat flour particle sizes on the physical properties, sensorial perception, and nutritional profile of the obtained bread. Therefore, the aim of this study was to make a complete characterization from a technological and a nutritional point of view of optimal wheat–buckwheat composite flours with large, medium, and small particle sizes, as established in our previously study. In addition, the bread features were evaluated. This work could contribute to the design of high-quality flour-based products enriched with different buckwheat particle sizes in the future.Saccharomyces cerevisiae yeast was purchased from Rompak company , and the salt and buckwheat seeds were bought at a market . The buckwheat composition consisted of total protein (13.26%), moisture (13.28%), fat (3.40%), and ash (2.00%) on a dry weight basis. Buckwheat seeds with coatings were milled and sieved according to our previous research [The wheat flour  was supplied by a bread factory , the fresh research ,32, to oIn our previous research  we deterBreads were made through the biphasic dough method, as previously described by Cotovanu et al.  from WF The moisture, ash, protein, and lipids, expressed as % (dry basis), were determined according to the International Association for Cereal Science and Technology (ICC) methods . The moiThe mineral contents, phosphorus (P), zinc (Zn), iron (Fe), calcium (Ca), magnesium (Mg), potassium (K), and sodium (Na) of the flour and bread samples were determined by using a flame atomic absorption spectrometry (FAAS)  equipped with air-acetylene flame, following our previous procedures ,36. The m/z, and a sampling rate of 3.5 scans/s [The sample (3.7 g flour or bread) was prepared in accordance with a method that had been previously published . The fil scans/s .max), and creep-recovery compliance .The determination of the dough dynamic rheological characteristics was performed by using a Thermo-HAAKE, MARS 40  with parallel plate–plate geometry (gap width of 4 mm), following Mironeasa et al.’s  method. The α-amylase activity was assessed with a Falling Number equipment , and the test also provided valuable information about starch grain quality .The flour thermo-mechanical properties were determined by using the Mixolab device , through the Chopin+ protocol, following the ICC 173 method . The theThe biaxial extension behavior of wheat/composite dough was evaluated by using a Chopin Alveograph NG-97  . The recm), total carbon dioxide volume production (VT), the volume of the gas retained in the dough at the end of the test (VR), and the gas retention coefficient (RC).The gaseous release and the batter development were assessed by using the Rheofermentometer device, type F4  . The reg3/g), and porosity and elasticity were calculated based on the Romanian standard [The bread weight was measured with an analytical balance. Bread volume (BV) was determined by the rapeseed displacement method and specific volume (Sp_Volume), expressed as the volume/weight ratio of bread  to white (100), value a* ranged from green (−) to red (+), and value b* varied from blue (−) to yellow (+).The bread color was measured at five opposite points around each sample with a chromameter  and expressed in the CIEA sensory evaluation of the loaves was performed by 13 semi-trained judges 2 h after cooling. The sensory analysis methodology used in this study was approved by the Ethical Committee of the Food Engineering Department of Stefan cel Mare University . The panr) was determined to identify correlations between the studied parameters.All the experiments were conducted at least in duplicate, and the data expressed as mean ± standard deviation. Statistical analyses were carried out using IBM SPSS Statistics 25.0  and XLSTAT 2021.2.1 software . The one-way analysis of variance (ANOVA) was applied to assess significant differences among samples at a 95% confidence interval. The multivariate data analysis technique, principal component analysis (PCA), was used to discriminate among samples and/or characteristics, and the Pearson correlation coefficient , compared to optimal composite wheat–buckwheat flours which ranged from 13.86 to 13.94% .p ˂ 0.05) higher than the one with large and small PS (OF_BL and OF_ BS) and WF. The lipid content varied from 1.44 to 1.86% in optimal composite flour with large (OF_BL), medium (OF_BM), and small (OF_BM) PS. Significant differences (p ˂ 0.05) regarding lipid content were obtained only on the OF_BM and the other two optimal composite flour samples. The ash content of optimal composite flours varied between 0.72 and 1.64%, which is higher than that of wheat flour (0.69%). The highest ash content (1.64%) was observed in OF_BM (p > 0.05) differences between the samples.The protein content of the optimal wheat–buckwheat composite flours varied between 12.00 and 14.14%, the highest percentage being present in the sample with medium PS (OF_BM) (14.14%), significantly , threonine (3.30–3.42%), tryptophan (12.39–12.59%), valine (4.40–17.50%), alanine (4.26–5.69%), aspartic acid (1.85–6.19%), glutamic acid (8.50–18.36%), glutamine (21.88–24.09%), glycine (6.12–7.75%), serine (7.24–8.23%), tyrosine (3.97–5.55%), and asparagine (6.18–6.85%). Following the trend of nutritional composition and mineral content, also in the case of amino acid content, the highest values were observed in the OF_BM.The application of numerical optimization in a previous study allowed the establishment of the optimal proportion of wheat:buckwheat in composite flour formulate for the large (90.87:9.13), medium (89.43:10.57), and small (89.75:10.25) buckwheat flour particle sizes, with the aim of obtaining a wheat–buckwheat composite flour and bread with the desired quality attributes . These oThe dough viscosity factor for the composite flour dough with the small particle size showed a remarkably higher value than the control, followed by the other optimal samples with large and medium particles. Overall, the optimal composite flour dough presented a decrease of creep-recovery compliance compared to the control.3) than the WFB volume (352.20 cm3). Bread volume increased with increasing buckwheat flour particle size in composite flour, and also the bread crumb porosity and elasticity were improved. The highest values of bread volume, porosity, and elasticity were registered in OB_BL .The optimal breads showed a significantly higher volume (362.51–373.19 cmL*), the intensity of the red (a*) or green (−a*) and the yellow (b*) for the optimal bread crust and crumb have varied depending on distinctive dose corresponding to the particle size dimensions (The lightness (mensions .a*) and yellow (b*) color intensity of the control bread crust was significantly lower than that of the optimal composite flour bread samples. Bread with the optimal dose of buckwheat flour addition that was distinctive for each particle size was marked by a significantly lower lightness of the crumb, within the range of 60.71–63.28, compared to wheat bread (73.94). The value of this parameter decreased significantly with the increase of the particle size. When buckwheat flour particle size decreased, a considerable rise in redness (a*) was observed, along with a lowering of yellowness (b*) in breadcrumbs, from 17.41 to 16.15. The a* value for the optimal bread crumb varied in the negative range (from −3.91 to −2.63), results which indicate that the bread crumb became darker and less yellow with the addition of buckwheat flour.As expected, the addition of buckwheat flour affected the color of the resulting loaves. The bread crust lightness decreased significantly in optimal breads with buckwheat flour compared to the control, with the OB_BM presenting the lowest value for this parameter. The value of the red  differences for firmness, springiness, gumminess, and masticability compared to the WFB. As illustrated in The optimal bread samples showed significant (p ˂ 0.05) both among them and compared to the WFB  being observed for OB_BM. The optimal bread samples have a raised protein content compared to the WFB. The sample OB_BM showed the highest protein content (10.00%), followed by the sample OB_BS (9.70%), and these results are correlated with the nutritional value of the buckwheat flour fractions . The lipThe essential and non-essential amino acid content of the optimal bread for each buckwheat flour fraction compared to the WFB showed variations depending on the size of the fraction a,b.Essential AA represents 14.60% for OB_BL, 12.20% for OB_BM, and 13.19% for OB_BS, of the total content of AA. The following variations in amino acids were registered in optimal breads: isoleucine (4.60–6.35%), leucine (4.20–5.88%), methionine (7.30–8.54%), phenylalanine (8.10–9.97%), threonine (7.46–8.87%), valine (4.40–7.87%), alanine (6.05–8.46%), aspartic acid (28.90–41.73%), glutamic acid (10.75–84.00%), glutamine (106.50–114.40%), glycine (3.25–10.05%), serine (6.00–7.65%), tyrosine (10.2–11.20%), asparagine (6.88–7.38%), proline (7.39–7.98%), thioproline (14.25–18.20%), and hydroxyproline (8.88–11.49%). An increase of essential amino acids was observed in the optimal bread compared to the WFB. Optimal breads with medium and small particle sizes were characterized by considerably higher amino acid content compared to WFB, especially leucine, methionine, threonine, valine, serine, glutamic, and aspartic acid.The mineral content varaition in the bread samples obtained  was assoA decrease in the content of macro-elements in optimal bread was observed with the decrease of the buckwheat flour particle sizes, while the content of micro-elements showed the following increase depending on the particle size: OB_BM > OB_BS > OB_BL a,b. RegaThe sensory evaluation results highlighted a considerable effect of the optimal buckwheat flour of large, medium, and small particle sizes which partially substituted refined wheat on the bread sensory quality and overall acceptability. The optimal bread with large and medium particle sizes (OB_BL and OB_BM) presented a higher score for sensory characteristics evaluated compared to wheat flour bread (WFB) .The optimal bread made with the small particles of buckwheat flour (OB_BS) which received a general acceptability score of 8.06, compared to the score of 8.35 received by the WFB, and 8.83 and 8.76 received for the OB_BL and OB_BM, respectively. Taking into account this information, it can be concluded that all optimal breads will probably be well accepted by potential consumers.Principal component analysis (PCA) allowed us to visualize the similarities and/or differences between the variables. It was observed that the first two components explained 86.12% of the total variance, where the first principal component (PC1) explained 57.86% .Optimal wheat–buckwheat composite flour showed proximate intermediate composition, based on the doses of buckwheat flour typical to particle sizes and to wheat flour. Similar results to those obtained in the present research regarding the protein content of optimal composite flour with particular particle sizes have been reported in some studies ,44,45. TTechnological behaviour differences occurred between optimal wheat–buckwheat flour samples and the control one. This phenomenon can be explained by the non-gluten matrix, fats, minerals, and sugars from the buckwheat flour particle sizes and their interactions , but alsThe reofermentometric indicators for all optimal wheat–buckwheat composite dough increased, a fact which can be explained by the fermentable carbohydrates from buckwheat flour and also by the biphasic method. Consequently, bread volume registered an increase in all samples, compared to WFB. This phenomenon may be related to the lower amount of gluten replaced, which will generate spatial structure modification of gliadin and glutenin molecular chains, with strong disulfide bonds that lead to inter- or intramolecular interactions of disulfide, ionic, or hydrogen bonds . Some auThe increase of G′ and G″ moduli was registered for all of the optimal samples compared to the WFB. This behaviour could be due to the limited plasticizing effect that favors gluten aggregation and determines a more elastic behavior of this latter sample. The highest increase in the visco-elastic moduli was registered in composite flour dough with a small particle size, variations that are probably influenced by the α-amylase amount of the optimal flour. The maximum gelatinization temperature dropped considerably compared to the control, the lower value being found for composite flour dough with a small buckwheat flour particle zise (OF_BS). The dough with a medium particle size of buckwheat flour (OF_BM) showed a viscosity factor and a maximum recovery compliance comparable to the values obtained for wheat flour dough.The improved properties of the buckwheat flour-based bread were impacted by the flour’s chemical composition. This phenomenon can be related to the biphasic method of bread preparation, as well, and can be due to the hydrolysis of buckwheat proteins. Similar results regarding bread supplementation with buckwheat flour were obtained by Diowksz and Sadowska . All optL*) parameter plays a key role in the bakery industry. It was shown that optimal breads presented a darker crust and crumb than wheat bread which can be explained by the increased fiber content from buckwheat flour. The lowest L* value (60.71) was obtained for the sample with the large particle size, suggesting a dark bread. Our results fall in line with those obtained by Psodorov et al. [The lightness  of the dough samples was positively correlated with the moisture of the bread samples (r = 0.99) but negatively correlated with the bread taste (r = −0.95). Indirect relationships were obtained between bread moisture and taste (r = −0.96). The lipid content of bread samples was positively correlated with bread volume (r = 0.95), but negatively correlated with bread gumminess and chewiness (r = −0.96). The ash content of the bread was positively associated with bread acceptability (r = 0.97). PC1 was associated with the maximum gelatinization temperature, the maximum creep and recovery compliance of the dough, and the content of proteins, lipids, ash, firmness, gumminess, volume, appearance, and smell of breads. It was observed that PC1 highlighted the opposition between the control bread and the OB_BM. The OB_BL showed a higher ash content, maximum recovery compliance, volume, porosity, taste, and acceptability, while the wheat bread showed high values for texture parameters and maximum gelatinization temperature. PC2 was associated with the elastic and viscous moduli, the viscosity factor (tan δ), and the moisture of the bread samples and crumb structure, and the highest values of these variables were obtained for the OB_BS.This work complements the previous study through complete characterization of the optimal wheat–buckwheat composite flours with large, medium, and small particle sizes established in there. Additionally, valuable information regarding the bread features was determined. The results showed an increase in volume, porosity, and elasticity for all loaves, whereas the firmness, crust, and crumb lightness decreased, compared to wheat bread. This trend is associated with the technological characteristics of optimal composite flours. Among all the optimal bread samples that with the medium particle (OB_BM) presents the highest protein and lipid content and had the best nutritional profile. Additionally, an improvement in amino acid content was obtained, but the highest values were registered for bread with small (OB_BS) and medium (OB_BM) particle sizes. Regarding the mineral profile of the bread samples, the highest content was obtained for that with large particle size (OB_BL), especially in terms of macro-element contents. The sensory evaluation highlighted that the panelists appreciated the breads made from optimal composite flours with large (OB_BL) and medium (OB_BM) particle sizes the most. These findings advocate for the use of wheat–buckwheat composite flour with large, medium, and small particle sizes to enhance the physical, textural, nutritional, and sensorial features of bread.


The article deals with increasing the mechanical properties of stainless steel 316 Ln-IG, which is intended for work in cryogenic temperatures (liquid nitrogen and liquid helium), such as conductor conduits for the ITER magnet system. The strength and plastic properties were increased by a combination of cold and cryo-rolling and heat treatment. The mechanical properties of rolled material were investigated at 293 K, 77 K, and 4.2 K. The work-hardening rate of the steel increased continuously with a lowering of the temperature. The maximum yield strength and ultimate tensile strength were achieved by the cryo-rolling process with a total thickness deformation of 50%. The material properties tested at ambient temperature were 0.2YS = 1050 MPa, UTS = 1200 MPa, and at 4.2 K, the values were 0.2YS = 1804 MPa and UTS = 2081 MPa. Two types of long-term heat treatment were applied after experimental rolling (823 K and 1093 K for 10 h). The highest precipitation hardening of steel was achieved at a temperature of 823 K after 50% deformation. The resulting grain size decreased from the initial 216 μm (before the rolling process) to 70 μm after ambient rolling and 72 μm after cryo-rolling. Due to its good corrosion resistance and malleability, austenitic stainless steel is one of the most attractive construction materials in the energy industry ,2,3. TheThe first information about the rolling of stainless steels under cryogenic conditions (CR) was published by Üçok  in 1991.The plastic deformation of stainless steels carried out at cryogenic temperatures leads to a remarkable increase in strength and hardness, but also to a decrease in elongation. The studies ,25 have 23C6 and M6C and intermetallic phases such as phase χ (Chi), σ (Sigma), and η (Laves) may be considered . The experimental rolling process was carried out in a four-high roll arrangement .Two types of experimental unidirectional rolling were carried out: conventional rolling at ambient temperature (AR) and rolling at cryogenic temperature (CR). Samples were rolled with about 10% reduction for each pass, and the total thickness deformation was Samples for DSC analysis were cut using the wire-cutting machine. The input weight of the samples was 20 mg. The DSC analysis was performed with a heating rate of 30 K/min in a nitrogen atmosphere using the simultaneous thermal analyzer, a STA 449 F1 Jupiter .After rolling, heat treatments As received material after solution annealing, it has the label SA. The system of sample labeling after rolling and heat treatment is shown in The static tensile test was carried out at TINIUS OLSEN H300KU  for ambient temperature and 77 K testing, and at MTS 100 Landmark , which was equipped with a cryostat and an extensometer. −1, which corresponds to a deformation speed of 0.00025 s−1. These values correspond to the standard for tensile testing [0 = 22 mm. At the ambient temperature and at 77 K for the static tensile tests, at least three tests were carried out for all deformed states and heat treatments. At a temperature of 4.2 K, there were two tests for each state.The samples for the static tensile test were cut from material in the longitudinal direction of the rolling process, as shown in  testing . The iniThe Vickers hardness test HV30 was carried out using the hardness tester HPO 250. The total load for testing was 294 N.3 = 3:1), and methanolic aquaregia diluted with methanol in a volume ratio of HCl: HNO3: methanol = 3:1:1. The etching time varied from a few seconds to minutes, depending on total deformation and heat treatment conditions.Cross-sectional samples for light optical microscopy (LOM) analysis were cut from rolled material parallel to the rolling direction with a saw with a water cooling system using Accutom 100 . The samples were ground and polished by standard procedures recommended by Struers, on the Tegramin 25 machine . Polished samples were etched in two different etchants: aquaregia solution, which contained hydrochloric acid and nitric acid in a volume ratio of 1:3 . Digital micrographs of 2560 × 1920 pixels were taken for each sample. The magnifications of 50×, 200×, and 500× were used. The micrographs were analyzed according to the standard for determining grain size . DSC curve are visible. The first peak is in the temperature range from 671 K to 874 K, which reveals multiple effects. In total, three peaks can be observed, with the main maximum at 784 K and two additional low maxima at about 750 K and 838 K. All these exothermic peaks can be caused by the precipitation of M23C6 type carbides. The point of formation of M23C6 (chromium-rich carbide) and intermetallic phases was mentioned in [23C6 precipitation. From the DSC curve, it is not possible to clearly attribute the temperatures to individual stages of precipitation. The development of precipitates can also be influenced by thermomechanical processing, while large plastic deformation significantly accelerates precipitation [23C6 precipitates. This effect was already observed at a temperature of 1123 K [The dependence of heat released for the stainless steel sample after solution annealing versus temperature during constant heating at a rate of 30 K/min is shown in ioned in ,33 at a ioned in , using tpitation . The secf 1123 K , represeTo create an optimal synergy between the structural and mechanical properties of structural stainless steel processed using plastic deformations, it is necessary to apply a suitable heat treatment. This must be done in such a way as to reconcile the mutual interaction between softening and precipitation hardening, possibly by recrystallization.Based on the hardness and the DSC measurements, two annealing temperatures for the steel processing for long-term annealing were determined: HT1 = 823 K and HT2 = 1093 K. The maximum increase in mechanical properties was expected at HT1. The heat treatment at HT2 was used to check the thermal stability of precipitation strengthening. To assess the mechanical properties of the fully recrystallized structure, the thermal treatment HT3 = 1323 K was determined and applied to the samples with the highest deformation (A50 and C50).SA,293K = 325 MPa, ultimate tensile strength UTSSA,293K = 641 MPa and total elongation TESA,293K = 49%. The ITER applications require a yield strength 0.2YSSA,293K = 220 MPa, ultimate tensile strength UTSSA,293K = 525 MPa, and total elongation TESA,293K = 45% [The initial state of 316LN-IG after solution annealing has more than 100 MPa higher mechanical properties at ambient temperature than is required for ITER applications with yield strength 0.2YSAt ambient temperature, the material shows a high supply of plasticity, where uniform elongation reaches 40% (UTS/0.2YS ≈ 2). At the same time, the most significant strain hardening rate is observed only up to deformation φ = 0.05, after which it gradually decreases.SA,77K = 790 MPa and ultimate tensile strength UTSSA,77K = 1282 MPa, and total elongation increased to TESA,77K = 57%. It is also possible to observe not only an increase in the uniform elongation UESA,77K = 50%, but also the strain hardening rate, especially up to the value φ = 0.15. These results confirm that the material ASS 316LN-IG, due to its fcc (face-centered cubic) structure, exhibits excellent deformability at cryogenic temperatures.At liquid nitrogen temperature, the material properties increased significantly, more than twice, where yield strength was 0.2YSIn the conditions of liquid helium, stress oscillations occur during a single-axis tensile test controlled by displacement, which creates the so-called discontinuous plastic flow (DPF). This phenomenon occurs when the temperature approaches absolute zero, below temperature T1, which for 316L is around 35 K . Two basSA,4.2K = 1070 MPa and ultimate tensile strength UTSSA,4.2K = 1543 MPa, while the total elongation was maintained at the same value, TESA,4.2K = 45%. The plastic flow in the initial stage shows a further increase in the strain hardening rate.The mechanical properties and strain hardening rate of the steel 316 LN-IG tested at 4.2 K were evaluated from the maximum values of plastic flow, which are shown in The results of the static tensile test of rolled material without any annealing process (HT0) are presented in C50%,4.2K = 2.2%.After the individual deformations in rolling, there is a significant strengthening of the material but also a dramatic reduction in plasticity when the total elongation, in some cases, reaches the value of TEA50,293K = 994 MPa, UTSA50,293K = 1055 MPa, with a minimum of TEA50,293K = 4.1%. Higher strength properties were achieved when the material was processed by cryo-rolling, with 50% of total deformation: 0.2YSC50,293K = 1050 MPa; UTSC50,293K = 1200 MPa; and TEC50,293K = 4.6%.The maximal level of strength properties of material processing by ambient rolling with 50% total deformation was obtained: 0.2YSIn the tensile test at ambient temperature, the material shows a higher 0.2YS after 10% and 30% deformations when rolling at cryogenic temperature. In comparison, after 50% deformations, the observed trend is the opposite. The lower 0.2YS at cryo-rolling (10% and 30%) is probably due to the mixed mechanism of plastic deformation (slip and twinning), which can lead to heterogeneity in the strengthening of the structure. After testing the material in cryogenic conditions (77 K and 4.2 K), it is observed that higher strengthening values (0.2YS and UTS) are achieved after rolling at cryogenic temperatures than after ambient rolling. This is probably because the low testing temperatures promote plastic deformation by twinning.C50,77K = 1571 MPa; UTSC50,77K = 1880 MPa; and a minimum of TEC50,77K = 3%. Mechanical properties  after ambient rolling with 50% deformation showed 9% lower values than after rolling in cryogenics.The maximal level of strength properties at the testing temperature of 77 K was obtained by cryo-rolling with 50% deformation: 0.2YSSA50,4.2K = 1755 MPa, UTSA50,4.2K = 1940 MPa, and a minimum of TEA50,4.2K = 7%. The higher strength properties were achieved when the material was processed by cryo-rolling at 4.2 K: 0.2YSC50,4.2K = 1804 MPa, UTSC50,4.2K = 2081 MPa, and a minimum of TEC50,4.2K = 2%.The curves from static tensile tests carried out at a temperature of 4.2 K on the samples after rolling at ambient and cryogenic temperatures are given in The initial state structure of 316LN austenitic stainless steel (referred to below as SA) is shown in The micrographs of the deformed structure after ambient and cryogenic rolling (without heat treatment) are shown in A significant number of deformation bands are formed in some grains, even at a strain of 30% thickness deformation. The detailed deformation bands and shear bands are shown in After the rolling at ambient temperature and ε = 50% (AR 50%) deformation, former grain boundaries and grains of the original size are slightly visible. A high number of deformation bands and low-angle grain boundaries are observed in all grains. More grains tilt in the rolling direction (a pancake-like shape).The grain size decreases with increasing levels of total thickness deformation ε. From solution-annealed grains with an average diameter of approx. 200 μm, the grain size has changed to 77 μm after 30% ambient rolling and 64 μm after cryo-rolling, respectively. In the case of 50% total-thickness deformation, an evaluation of grain size is slightly complicated, due to the strong deformation of the microstructure, and grain boundaries are no longer clearly visible. The deformation is inhomogeneous. The grains are elongated in the rolling direction. In the direction of the rolling thickness (ND), the grains have dimensions from 10 to 200 µm. In between these huge, elongated grains, the strongly deformed microstructure, made up of tangles of deformation bands, shear bands, and twins, was observed. The small grains without well-defined boundaries have a diameter of 50 μm or less.A detailed description of the mutual interaction between the shear bands and deformation bands is shown in Heat treatment according to conditions HT1 and HT2 was carried out on samples that were rolled at ambient temperature and in liquid nitrogen with deformations of 10, 30, and 50%. The samples of the HT3 state were deformed by 50% (AR and CR).C50,293K,HT1 = 1230 MPa, UTSC50,293K,HT1 = 1315 MPa, and TEC50,293K,HT1 = 7.4%. This condition when tested in liquid nitrogen also achieved a significant increase in mechanical properties, where 0.2YSC50,77K,HT1 = 1735 MPa, UTSC50,77K,HT1 = 1895 MPa, and TEC50,77K,HT1 = 11.4%.The development of mechanical properties after HT1 is shown in C50,77K,HT2 = 1135 MPa, UTSC50,77K,HT2 = 1475 MPa, and TEC50,77K,HT2 = 14%.The heat treatment HT2 caused a sharp decrease in strength properties, mainly for states after 30% and 50% deformations (AR and CR). All deformation states show very similar strength properties at ambient temperature, where YS ≈ 590 MPa and UTS ≈ 800 MPa . Under tA50,77K,HT3 = 377 MPa, UTSA50,77K,HT3 = 669 MPa, and TEA50,77K,HT3 = 55%, while these values are noticeably higher after CR. Even when tested in cryogenic conditions, the material shows lower strength properties than the base material.The heat treatment HT3 was carried out to determine the mechanical properties in cases where the temperature of the material reaches the recrystallization temperature. The mechanical properties of stainless steel 316LN-IG after 50% deformation  followed by solid solution annealing (HT3) are shown in In The annealing conditions of HT2 evidently reduce the strength properties  are shown in The micrographs of the deformed structure after ambient and cryogenic rolling followed by heat treatment at 823 K for 10 h are shown in The micrographs of the deformed structure after ambient and cryogenic rolling followed by heat treatment at 1093 K for 10 h are shown in The grain size of ambient AS rolled by 30% and heat-treated AS at 823 K is 55 μm for ambient rolling and 9 μm for cryo-rolling. The difference between grain sizes may be caused by a partial recovery process or, in the case of cryogenically rolled material, by the new grains. These small grains reduce the average grain diameter rapidly. The material cryo-rolled by 30% and the heat-treated SA at 1093 K have an average grain diameter of approx. 10 μm for both types of the rolling process. The difference in microstructure is in the LAGB/HAGB ratio and the ratio of annealing twins. This will be examined through further analysis. The comparison of the microstructure of AR 50% after HT1 and HT2 is shown in According to the lower temperature of HT1, partial recovery is obvious, but the material is stored at a high level of strain. After HT2, new austenite grains with an average diameter of 3–5 μm and precipitates are visible b.The completely recrystallized austenitic structure was observed after 50% deformation and following heat treatment at 1323 K for 1.5 h . Some an293K = 641 MPa, UTS77K = 1282 MPa, and UTS4.2K = 1543 MPa). Even at very low temperatures, the material shows high ductility TESA,77K = 57% and TESA,4.2K = 45%.Austenitic stainless steel 316 LN-IG, after solid solution annealing, shows excellent synergy between strength and plastic properties at cryogenic temperatures. The strength of the steel increases continuously with decreasing testing temperatures  after 50% deformation.At an annealing temperature of 1093 K, there was a loss of deformation and precipitation hardening in the steel.The completely recrystallized austenitic structure was observed after 50% deformation and following heat treatment at 1323 K for 1.5 h.Tensile properties of austenitic stainless steel 316 LN-IG were investigated in the temperature range 293–4.2 K under different conditions of heat-deformation treatment. Mechanical properties after rolling at ambient temperature and in liquid nitrogen, as well as after subsequent heat treatment, were compared. Individual stages of thermomechanical processing were analyzed using optical microscopy. The main results are summarized as follows:


Knowledge of aquifer dynamics, including groundwater storage changes, is key to effective groundwater resource and reservoir management. Resolving and accurate modeling of these processes requires knowledge of subsurface poroelastic properties and lateral heterogeneity within units of interest. Computationally demanding methods for determining lateral heterogeneity in poroelastic properties exist but remain difficult to practically employ. The InSAR-based detection of uplift over a New Mexico well with a casing breach provides an opportunity to determine poroelastic properties using a tractable 2D analytical plane strain solution for surface uplift created by a pressurized reservoir with overburden. Using a Bayesian inversion framework, we calculate poroelastic properties under deep (depth of well-screen) and shallow (depth of well-breach) conditions. We find that shallow injection is necessary to produce the observed deformation. However, pressure-varying forward solutions for uplift are required to reproduce the temporal evolution of deformation. For this we use realistic shallow poroelastic properties and well dynamics, which reflect the evolving injection conditions at the well breach as the casing further erodes. Analysis of individual interferograms or InSAR time series may provide insights into shallow subsurface heterogeneity or anomalous injection conditions at operating wells more rapidly than scheduled field inspections. These observations help us understand injection impacts on hazards such as aquifer pollution, halite dissolution and sinkhole formation9, as well as the potential for induced seismicity10. They are also important for groundwater resource and reservoir management, monitoring, and sustainability as surface deformation provides insight into the temporal and spatial dynamics of aquifer storage capacity and a way to observe unrecoverable aquifer storage loss12.Large-scale deformation caused by groundwater pumping and oil and gas production has been identified using InSAR14. The lateral boundaries of productive regions often can only be defined at the level of ‘between this well and that well’. Productive fluvial and alluvial formations that commonly house freshwater aquifers are especially heterogeneous, with lateral boundaries sometimes appearing nearly randomly and at short length scales14. The ability to map hydraulic properties without expensive, indirect geophysics or prohibitively expensive drilling programs has remained a grand challenge in hydrology from the first uses of groundwater16. Efforts towards using InSAR to characterize lateral heterogeneity include the joint analysis of InSAR and hydraulic head measurements taken at observation wells21 or production wells12 to derive hydromechanical properties  and the identification of new faults disrupting groundwater flow22. However, the analysis of sub-kilometer scale deformation in wellfields to gain insight into poroelastic properties has been largely overlooked.Understanding subsurface properties and their lateral heterogeneity is another key factor in the management and monitoring of reservoirs. In most cases, aquifers are developed through trial-and-error drilling with limited geophysical surveys, though this is changing with evolving technologies23 use a computationally expensive Bayesian inversion method for InSAR observations over a wellsite in Nevada to find variations in lateral permeability, inferring up to ~ 17,000 permeability parameters. The authors use a 3-D geomechanical model with over 320,000 pressure, displacement, and fluid flux state variables. The complexity and computational cost of running such an extensive inversion, however, makes implementation prohibitive for many practical applications.Alghamdi et al.24 describe one example of such sub-kilometer scale deformation at a wastewater injection well in West Texas attributed to casing failure and/or sealing problems and propose utilizing InSAR as an indirect leakage monitoring method. Our findings support this proposed method and illustrate its effectiveness compared to scheduled inspections.Here, using InSAR time series, we resolve an injection plume that propagates outward from a well in southeastern New Mexico’s Vacuum wellfield in the Permian Basin Fig.  startingOur resolved time series of deformation reveals an initially nearly circular deformation pattern that over time develops a linear asymmetry, which we believe is part of a subsurface lateral heterogeneity in shallow formations. To understand which formation parameters are most reflected in deformation in a homogeneous formation, we determine poroelastic properties of the subsurface from the data utilizing a simple 2-layer analytical model coupling a 1D groundwater flow equation with a plane strain solution for surface uplift due to a pressurized reservoir with overburden. This essentially controlled shallow injection experiment demonstrates the value of high-resolution remotely sensed surface deformation time series in imaging and characterizing lateral heterogeneities either in structure or lithology of groundwater bearing formations with direct implications on the modeling of groundwater storage and aquifer dynamics. Importantly, it suggests that deformation data can be used in some cases to determine aquifer properties – hydraulic diffusivity, formation thickness, lateral boundaries – without the need for a dense monitoring well network. This also provides a method for well field monitoring that can be more effective and timely in its response than traditional manual inspections.25 on ascending path 78 frame 104 and descending path 85 frame 484 from October 2016 through April 2019  and many display high coherence and minimal short wavelength atmospheric noise Fig. . Because31 to create a 2D analytical model of the injection conditions and lithology at wellsite American Petroleum Institute (API) No. 3002524312. Modeling reservoir pressure with a 1D, transient solution to the groundwater flow equation for an isotropic and homogeneous formation, we set no-flow boundaries at the top and bottom of the aquifer, an infinite right-hand domain, and a fixed pressure boundary on the left-hand side (the location of the wellbore). We assume a plane-strain approximation for modeling geomechanics and couple the equations via standard poroelasticity through the Biot coefficient to tie effective stress and overpressure to each other. We assume the formation elastically deforms much faster than the pressure wave propagation which allows a one-way coupling from the groundwater flow equation to the plane strain deformation solution.We follow the methods outlined by Wangen et al.h1, and an overburden of thickness h2. The lateral domain extends from -L to L. Boundary conditions include the absence of vertical displacement at the base of the reservoir, the overburden surface experiences zero normal stress, and the pressure distribution, p(x), is symmetric about the wellbore or z-axis at x = 0 and reaches zero before the domain ends at |x|= L.This plane strain solution, derived from a Fourier decomposition of the reservoir overpressure, determines the surface uplift over a pressurized reservoir and an additional layer of overburden. This simple two-layer model contains a reservoir with a thickness, 31, we determine the surface uplift, p0, using Eq.  represents the overpressure at time t and location x given an initial fluid pressure p0 and hydraulic diffusivity D. Assuming uniform hydraulic diffusivity and solving the groundwater conservation equation in 2D, we approximate the pressure wave propagation with.32.We find the Fourier coefficients, ting Eq. 4\documen33. Conversion of the well from oil production to water injection occurred in the early 1980’s. An injection profile survey reported on 14 Aug. 2017, resulted in the shut in of the well following the identification of a casing leak between 256 and 265 m. All attempts to repair the well failed, and after once again finding material from the surrounding Dewey Lake Formation redbeds in the wellbore, the well was plugged and by 01 Sep. 2017 the site was abandoned. To resolve the source of deformation, we create a model for the intended deep injection into the San Andres formation at 1463 m depth, and another for shallow injection occurring at the well breach between 256 and 265 m.We model two separate conditions at the wellsite to account for the well’s history according to the State of New Mexico Oil Conservation Division (NM-OCD) which provides details of operational conditions, injection / production, and maintenance on individual wellsites including those located within the Vacuum wellfield. From their reports we know Texaco Inc. began drilling API well No. 3002524312 in December 1972, and reached a total depth of 1463 m before production started the following January 197334. Largely dolomite, the reservoir also includes sections of impermeable anhydrite cementation and very low permeability thin, interbedded dolomitic siltstones34, which also may compartmentalize the reservoir. Overlying Guadalupian formations of dolomite and anhydrite include secondary shale and sandstone layers. Younger formations are predominantly composed of dolomite, anhydrite, sandstone, and halite34. The youngest formation, the Late Permian Dewey Lake Formation, consists of anhydrite cemented fine sandstone and siltstone35. Schiel36 attributes the geomorphologic features and sedimentology of the formation to deposition in an ephemeral fluvial system featuring sheet floods, and broad, shallow channels up to 2 m thick that stack and crosscut one another. Above the Dewey Lake Formation lies the Triassic-aged Santa Rosa Formation (Dockum Group), a complex body of sandstones with channels up to 30 m thick that have been found to be permeable across the region37. Other channel sand bodies have been found in the Dockum Group above the basal Santa Rosa Formation as well38 and are anecdotally described as ‘pockets of water’; however, due to poor water quality in this region, the Dewey Lake Formation sand bodies are relatively unexplored.The Guadalupian-aged Grayburg and San Andres formations form the primarily carbonate reservoir of the Vacuum wellfield. High-angle, low-displacement faults horizontally compartmentalize the reservoir39 that implements Bayesian inversions via a Markov Chain Monte Carlo (MCMC) method to infer posterior probability density distributions of values for free model parameters: depth to reservoir/overburden thickness, width of reservoir, borehole pressure, diffusivity, and Young’s modulus. We begin with uniform prior distributions using parameter bounds accounting for the well geometry and lithology of the reservoir and overburden during deep injection  and compare the values of uplift to observed InSAR uplift profiles Fig. .Setting the depth of injection to the range where perforations were made in the lowest section on the well with an upper bound of 1000 m in the prior uniform distribution, we invert the deformation observations for the model parameters reservoir depth and thickness, borehole pressure, diffusivity, and Young’s modulus. The posterior distributions in Fig. –4 m2/s) is physically reasonable for the rock type. The deformation resulting from the unrealistic MAP values for the poroelastic parameters significantly underestimates the observed deformation reaching a maximum of ~ 20 mm  value for depth is just below the imposed 1000 m limit, while the reservoir thickness of ~ 5 m and a Young’s modulus of ~ 2 GPa are not realistic values for characterizing the deep injection conditions and lithology. The MAP borehole pressure of ~ 92 MPa is also significantly above the reported operational conditions (10—15 MPa). The value for diffusivity  and a depth or thickness of overburden (h2) of ~ 186 m. The value of reservoir depth or overburden thickness is not consistent with NM-OCD reported values. MAP values for a diffusivity of ~ 1 × 10–3 m2/s and a Young’s modulus of ~ 8 GPa are reasonable values for the known geology, but a borehole pressure of ~ 20 MPa falls slightly outside of the reported operational range of values. The surface deformation for time t1-t6 given the MAP parameter values is plotted in Fig. Historical well injection data from the NM-OCD documents the presence of a casing breach somewhere between 256 and 265 mWe observe a trade off between values of depth and reservoir width where a deeper reservoir corresponds to a smaller reservoir thickness which allows for higher pressures in the reservoir. A narrow posterior distribution for diffusivity shows sensitivity for determination when using our methods, while a broad distribution for the Young’s modulus and borehole pressure reveals that they are not as well determined. Given the generally non-Gaussian distributions, we do not report the formal standard deviations of the parameter value distributions.40. Consistent with the image well solution, a clear linear feature develops along this barrier and deformation is noticeably more extensive in the southeast. We expect fluid migration to be altered by changes in permeability/lateral heterogeneity in the formation; therefore, preferential deformation to the southeast of this feature allows us to infer higher permeability in this region. The observed changes in poroelastic properties could be attributed to the stacked and cross-cutting fluvial channels of the Dewey Lake Formation.InSAR observations over southeastern New Mexico that span more than three years, the limit of coherence in this Sentinel-1 frame, document the temporal evolution of an injection-driven ground deformation signal. The study of individual interferograms Fig.  provides41; however, the formation depth limits the effect on the signal even with very large (1–3 orders of magnitude) changes in permeability. Two, the Dewey Lake Formation sand bodies, which are Santa Rosa Sandstone-equivalent sand bodies (Dockum Group), where we believe the leak occurred, generally have little clay and have undergone extensive lithification46, limiting the coupling. Three, the mean stress dependence of permeability in lithified materials at the depths of the San Andres Formation and Dockum Group generally results in permeability changes on the order of 10% (mean stress changes of 10 s MPa) which would exceed the ‘fracture gradient’47 and the casing leak would have likely been larger and apparent at the surface.Determining poroelastic properties for the region surrounding the wellsite becomes difficult when the assumed homogeneous overburden in our model is known to be heterogeneous . Model outputs for the two cases, injection at depth and shallow injection, indicate that shallow injection would be necessary to produce the uplift observed via InSAR at the reported injection rates. This shallow injection would have been possible given the NM-OCD reported well breach. Therefore, injection of fluid and gas likely occurred at shallower depths than intended and caused the measurable expression of uplift over the wellsite similar to that observed by Kim et al.8.Despite dense active injection well spacing of 0.04 kmIt is worth noting that the compromised well casing or otherwise anomalous well conditions could have been reported after acquisition of the first interferometric pair in Fig. 8), demonstrating the importance of timely well integrity observations.During the time between casing failure and the eventual halt of well operations, water unsaturated in NaCl infiltrated the rock surrounding the breach. Had injection occurred in a salt rich formation, such as the Salado Fm. at ~ 472 m depth, about 220 m below the casing breach, dissolution of the halite could lead to karst formation and eventual collapse (e.g., Wink Sinks24 also describe an example of sub-kilometer scale deformation at a wastewater injection well in West Texas. The signal was attributed to casing failure and/or sealing problems and they proposed utilizing InSAR as an indirect leakage monitoring method. Our results show another example of InSAR’s suitability to be used for this purpose. We not only provide a unique opportunistic example comparing the modeled depth to documented well casing damage for model verification, but show how these signals can be utilized to image lateral heterogeneity and characterize the shallow subsurface and well conditions through a simple modeling approach.Zheng et al.31 plane strain analytical model illustrates a low computational cost method of opportunistically determining poroelastic properties from the surface deformation at a wellsite. Whether shallow injection is conducted in a controlled manner, as in an aquifer test, or unintentionally as the result of mechanical failure as in our case, it can be utilized as a way to estimate hydraulic and poroelastic properties and to map local lateral formation heterogeneity.Using both forward and inverse models, we have shown that the InSAR observed deformation at an injection well can be attributed to the shallow injection of fluids due to a casing breach documented months after the first measurable deformation. This provides further evidence for the usefulness of InSAR-based leakage monitoring and the ability to accurately determine injection depth, while our application of Wangen et al.’s31 model can be used in a formal, relatively unconstrained Bayesian inversion to determine the effective/average diffusivity and to some degree the Young’s modulus; however, mechanical failures likely violate our assumptions of constant pressure resulting in mis-estimates of the parameters, thus showing that modeling results of well deformation have to be treated with caution. Simple pressure-varying forward models provide an approximated solution to this issue producing smaller residuals and realistic poroelastic properties and well geometries. The straightforward nature of these forward models makes analysis more approachable and time efficient for use in aquifer management and monitoring as well as oil and gas production, where early detection of casing breaches and sealing issues could prolong the life of wells, and prevent contamination or dissolution of the surrounding material and groundwater.Wangen et al.’sSupplementary Information.


Background: Although pulmonary vein isolation (PVI) is an established procedure for atrial fibrillation (AF), non-PV foci play a crucial role in AF recurrence. Persistent left superior vena cava (PLSVC) has been reported as critical non-PV foci. However, the effectiveness of provocation of AF triggers from PLSVC remains unclear. This study was designed to validate the usefulness of provoking AF triggers from PLSVC. Methods: This multicenter retrospective study included 37 patients with AF and PLSVC. To provoke triggers, AF was cardioverted, and re-initiation of AF was monitored under high-dose isoproterenol infusion. The patients were divided into two groups: those whose PLSVC had arrhythmogenic triggers initiating AF (Group A) and those whose PLSVC did not have triggers (Group B). Group A underwent isolation of PLSVC after PVI. Group B received PVI only. Results: Group A had 14 patients, whereas Group B had 23 patients. After a 3-year follow-up, no difference in the success rate for maintaining sinus rhythm was observed between the two groups. Group A was significantly younger and had lower CHADS2-VASc scores than Group B. Conclusions: The provocation of arrhythmogenic triggers from PLSVC was effective for the ablation strategy. PLSVC electrical isolation would not be necessary if arrhythmogenic triggers are not provoked. Because several studies have demonstrated that pulmonary veins (PVs) are the primary site of ectopic beats initiating atrial fibrillation (AF), PV isolation (PVI) has been established as a cornerstone ablation procedure for curing AF ,2. In coThis multicenter retrospective study included 37 patients with PLSVC from three centers who underwent catheter ablation for symptomatic drug-refractory AF from January 2005 to December 2019. The study protocol was approved by the Institution Research and Ethics Committee of all involved institutions and conducted according to the Declaration of Helsinki. Demographic data, including patient, background, procedural details, computed tomography (CT) imaging studies, follow-up visits, and echocardiograms were obtained from the medical records. All patients underwent preprocedural imaging using 64-slice contrast CT scanning  to evaluate the presence of the PLSVC and coexisting heart disease. All anti-arrhythmic drugs (AADs) were discontinued for at least five half-lives before the electrophysiological test and ablation procedure except for amiodarone, which was discontinued at least 7 days before ablation. A transesophageal echocardiogram was performed to exclude left atrial thrombus and investigate the other congenital heart disease. The procedure of PVI has been previously described in detail . PVI wasFollowing the PVI procedure, the electrophysiological study was performed in all patients to provoke the triggered activity from the PLSVC . VenograThe electrical isolation of the PLSVC was performed if the PLSVC had AF triggers to prevent the arrhythmogenic triggered activity from conducting to the atria. Before completing the electrical isolation of the PLSVC, premature ectopy with the earliest activation in the PLSVC was evaluated during the mapping procedure. If the location of the AF trigger emerged from the distal-PLSVC, electrical isolation of the PLSVC was achieved through a circumferential ablation at the level of the mid-PLSVC (between the left superior pulmonary vein and inferior pulmonary veins). If the location of the AF trigger was in the proximal-PLSVC, focal ablation or segmental isolation was performed targeting the trigger site of the PLSVC and LA connection. The power used for ablation in the PLSVC was 15–20 W and was restricted to a maximum time of 20 s for each ablation point with a flow rate of 17 mL/min, targeting a maximum temperature of 43 degrees. Successful isolation of PLSVC was defined as the achievement of either exit and entrance blocks or electrical disconnection between the PLSVC and LA, which was confirmed by the reversal of the activation sequence in the CS during LA appendage pacing. If disconnecting the electrical connection between the PLSVC and LA was difficult, applications from the LA were added as needed. Maximal output pacing through the ablation catheter was delivered to confirm left phrenic nerve was captured, but the point was not ablated. After the ablation to the PLSVC, a high-dose isoproterenol infusion was performed to verify that all triggers were isolated or eliminated. All patients visited the clinic and underwent 12-lead electrocardiogram recording 1, 3, 6, 12, 15, 18, 21, 24, 30, and 36 months after the procedure. All patients were equipped with an event recorder or 24 h electrocardiogram to analyze symptoms suggestive of arrhythmia recurrence. Antiarrhythmic drugs were continued for 3 months during the blanking period. Any documented episodes of atrial tachyarrhythmia lasting >30 s after 3 months of the blanking period were considered a recurrent arrhythmic event (late recurrence)—the occurrence of any serious adverse events within the first 30 days after the procedure was recorded. A standard definition of serious adverse events was used: any ablation- or procedure-related untoward medical occurrences leading to death or a deterioration in the patient’s health that resulted in life-threatening illness or injury, permanent impairment of a body structure or a body function, and/or prolonged inpatient hospitalization or medical or surgical intervention to prevent life-threatening illness or injury or permanent impairment to a body structure or a body function. t-test, the chi-square test, Fisher’s exact test, and the Kalan-Meier curve analysis with a long-rank test as appropriate, using Statistical Package for the Social Sciences, version 24.0 . p-value equal to or <0.05 was used to denote statistical significance.The data are presented as means ± standard deviations. The data were compared using Student’s In this study, 37 patients who had PLSVC with symptomatic drug-refractory AF who underwent catheter ablation were included. Patient characteristics are shown in p < 0.001) and had significantly lower CHA2DS2-VASc scores  (p < 0.001). The occurrence of any complications did not differ between the two groups.Electrical PVI was complete for all patients. Following the PVI procedure, all 37 patients underwent the provocation of arrhythmogenic triggers from the PLSVC. Through electrophysiological examination, 14 patients were determined to have AF triggers from the PLSVC (Group A), whereas 23 had no remarkable AF triggers from the PLSVC (Group B). The comparison of the two groups showed no difference in the duration of AF, left ventricular ejection fraction, and the diameter of the PLSVC. However, the patients in Group A were significantly younger  . The proAll 14 patients with AF triggers from the PLSVC underwent ablation procedures to the PLSVC. The triggers appeared from the distal PLSVC in 11 patients (81.3%) and from the proximal PLSVC in three patients (18.7%). The 11 patients who had AF triggers from the distal-PLSVC underwent electrical isolation at the level of the mid-PLSVC by circumferential applications. The three patients with AF trigger from the proximal-PLSVC underwent focal ablation or segmental isolation to the trigger site. No significant difference in the success rate of PLSVC isolation was observed; however, the procedure time was significantly longer in patients with AF trigger emerging from the proximal-PLSVC. All three patients with AF trigger from the proximal-PLSVC required additional ablation to the LA to disconnect the electrical connection between the PLSVC and LA . No compp = 0.607)  .The primary findings of this study are as follows: (a) the provocation and localization of the triggered activity initiating AF from the PLSVC were important to determine the ablation strategy; (b) the electrical isolation of the PLSVC, which had AF triggers associated with long-term freedom from AF at the 3-year follow up; (c) trigger-free PLSVC may not require electrical isolation; (d) the electrical isolation of the PLSVC was performed safely without serious complications; and (d) the location of the arrhythmogenic triggers in the PLSVC was important. If the arrhythmogenic triggers emerge from the proximal PLSVC, the electrical isolation of the PLSVC was not simple. Further ablations might be required from the LA because of the disconnection of various electrical connections between the PLSVC and LA. The role of the PLSVC in initiating AF was first identified by Hsu et al. , and theA prior study has reported that the electrical isolation of the PLSVC was associated with increased complications, such as cardiac tamponade . TherefoThere are several reasons for the arrhythmogenicity of the PLSVC . In the In this study, the electrical isolation of the PLSVC was performed safely, and no increased complications associated with the procedure were observed. If the provoked arrhythmogenic triggers emerged from the distal-PLSVC, the electrical isolation procedure of the PLSVC was relatively simple. Circumferential ablation to the PLSVC at the level between the left superior PV and left inferior PV was performed the same as right SVC electrical isolation. However, if the triggers emerged from the proximal-PLSVC, the isolation of the PLSVC was not straightforward because the paroxysmal-PLSVC has various electrical connections between the LA and PLSVC . FurtherThis study has several limitations. First, it was a non-randomized observational retrospective study with a small sample size. Second, the results were pooled from three centers and multiple operators with differences in the extent of ablation performed. However, the protocol for the provocation of arrhythmogenic triggers of non-PV foci was similar, and no significant difference in the 3-year outcomes of AF-free survival was observed between the two groups. Finally, we cannot rule out asymptomatic subclinical AF recurrence in patients as none of them had a cardiac implantable electronic device. Furthermore, we cannot rule out the possibility of having a more significant arrhythmia recurrence on longer follow-ups. The role of supplementally ablation procedures remains controversial and needs further investigation in ablation technology and patient selection.In patients with AF and PLSVC, the provocation and localization of the ectopic triggers from the PLSVC were effective for catheter ablation. PLSVC electrical isolation would not be necessary if the arrhythmogenic triggers were not provoked.


Nonalcoholic fatty liver disease (NAFLD) is substantiatedby thereprogramming of liver metabolic pathways that disrupts the homeostasisof lipid and glucose metabolism and thus promotes the progressionof the disease. The metabolic pathways associated with NAFLD are regulatedat different levels from gene transcription to various post-translationalmodifications including ubiquitination. Here, we used a novel orthogonalubiquitin transfer platform to identify pyruvate dehydrogenase A1(PDHA1) and acetyl-CoA acetyltransferase 1 (ACAT1), two importantenzymes that regulate glycolysis and ketogenesis, as substrates ofE3 ubiquitin ligase UBE3A/E6AP. We found that overexpression of UBE3Aaccelerated the degradation of PDHA1 and promoted glycolytic activitiesin HEK293 cells. Furthermore, a high-fat diet suppressed the expressionof UBE3A in the mouse liver, which was associated with increased ACAT1protein levels, while forced expression of UBE3A in the mouse liverresulted in decreased ACAT1 protein contents. As a result, the micewith forced expression of UBE3A in the liver exhibited enhanced accumulationof triglycerides, cholesterol, and ketone bodies. These results revealthe role of UBE3A in NAFLD development by inducing the degradationof ACAT1 in the liver and promoting lipid storage. Overall, our workuncovers an important mechanism underlying the regulation of glycolysisand lipid metabolism through UBE3A-mediated ubiquitination of PDHA1and ACAT1 to regulate their stabilities and enzymatic activities inthe cell. The nutrient metabolism in the liver is regulatedat different levels from gene transcription to the control of enzymaticactivities by allosteric ligands and post-translational modifications.For one, the transcriptional regulation of metabolic pathways in theliver is exquisitely mediated by a number of hormones such as insulin,glucagon, glucocorticoids, and so forth, which often perform counter-regulatoryfunctions. They cooperate to maintain the proper expression of enzymesand transporters involved in nutrient metabolism via switching onor off signaling cascades that affect the activities of transcriptionalfactors and the expression of their target genes.Ube3a gene is paternally inactivated in the brain, so itsUB ligase activity is vulnerable to mutations in the maternal copyof the gene that may render the gene defective and cause the delayof neuronal and intellectual development in children.30 On the other hand, the duplication of the Ube3a gene in the maternal chromosome is associated with autism spectrumdisorders.32Ube3a is normallyexpressed from both paternal and maternal copies of the chromosomein the peripheral tissues. The binding of the E6 protein of the humanpapillomavirus (HPV) with UBE3A would induce the degradation of thetumor suppressor p53, which is causative for the development of cervicalcancer.34 UBE3A has been recently shown to play arole in hepatic steatosis.35 In this study,we identified two essential enzymes regulating glycolysis, the TCAcycle, and ketogenesis, namely pyruvate dehydrogenase A1 (PDHA1) andacetyl-CoA acetyltransferase 1 (ACAT1), as ubiquitination targetsof UBE3A. We found that UBE3A regulates the stability and enzymaticactivity of PDHA1 and ACAT1 in the cell, and the overexpression ofUBE3A in the mouse liver would enhance glycolysis and ketogenesisto condition the liver for lipid accumulation as a primer for NAFLD.Our findings unveiled a new axis enabling the regulation of glucoseand lipid metabolism by protein ubiquitination and suggested a potentialrole for UBE3A in NAFLD development.UBE3A, also known as E6AP forE6-associated protein, is an E3 UBligase implicated in the development of cervical cancer and neurodegenerationsuch as Angelman syndrome. Due to genomic imprinting, the The following expression plasmids were used:the pET-28a plasmid for protein expression was from Novagen . pET-PDHA1 and pET-ACAT1 were generated by the insertionof full-length PDHA1 and ACAT1 genes cloned from HEK293 cDNA intothe pET-28a vector. The plvx-IRES-mCherry vector was provided by FengQian of Shanghai Jiao Tong University. plvx-UBE3A-IRES was generatedby the insertion of the full-length UBE3A gene cloned from HEK293cDNA into the plvx-IRES-mCherry vector. The pCAG/GFP plasmid (139980)was purchased from Addgene . pCAG-UBE3A was generatedby replacing the GFP gene between the BamHI and HindIII restrictionsites in the pCAG-GFP vector with the UBE3A gene. The GIPZ non-silencinglentiviral shRNA control (shControl) plasmid (RHS4348) and GIPZ shUBE3Aplasmids (#1–6)  were purchased from Horizon Discovery .The following antibodies were purchasedfrom Santa Cruz Biotechnology : anti-PDHA1 (sc-377092),anti-UBE3A (sc-166689), anti-actin (sc-8432), anti-UB (sc-8017), andmouse anti-rabbit IgG-HRP (sc-2357). The goat anti-mouse IgG secondaryantibody (31438) was from Thermo Fisher Scientific .Anti-ACAT1 (ab168342) was from Abcam .Escherichia coli BL21 (DE3) cellsharboring the expression constructs were cultured at 37 °C and200 rpm until they reached the exponential phase (OD600 = 0.6–0.8) and were induced with 1 mM IPTG at 16 °Cfor 20 h. Cells were harvested by centrifugation. Cell pellets wereresuspended in lysis buffer , followed by sonication to complete the lysis process. Thelysates were centrifuged, and the supernatant was incubated with Ni-NTAresin  and rocked gently at 4 °C for2 h. The protein-bound resin was loaded onto a QIAGEN column, washedtwice, and eluted with 250 mM imidazole. Purified proteins were analyzedby SDS-PAGE. The SDS-PAGE gel was stained in a Coomassie R-250 solutionfor 3 h, followed by distaining.2 and 1.5 mMATP, pH 7.4), including 5 μM of substrates PDHA1 (UniProt accessionID: P08559) or ACAT1 (UniProt accession ID: P24752), 1 μM Uba1 (UniProt accessionID: P22314), 5 μM UbcH7 (UniProt accession ID: P68036), 5 μMUBE3A (UniProt accession ID: Q05086), and 20 μM UB (UniProt accessionID: P62975). The reactions were incubated at 37 °C for 2 h, followed byboiling in SDS-PAGE loading buffer. The samples were analyzed by westernblotting that was probed with anti-substrate antibodies.The in vitro ubiquitinationassay was performed in 50 μL TBS  with 10% fetal bovine serum. Transienttransfection of cells was performed with the transfection reagentfrom Horizon Discovery , followingthe manufacturer’s instructions.Lentiviralparticles for the transduction of the shControl plasmid were preparedaccording to the manufacturer’s protocol for the Trans-LentiviralshRNA Packaging System. Briefly, the trans-lentiviral packaging plasmidmix (Dharmacon TLP5912) was co-transfected with the shControl plasmidinto HEK293T as the packaging cell line for the production of thelentiviral supernatant. The shControl lentiviruses obtained were thenused to infect HEK293 cells. The infected cells were seeded into cultureplates 48 h post-infection and cultured in cell medium containing1 μg/mL puromycin for screening stable shControl cells. Thegeneration of the stable shControl cell line was confirmed by theco-expression of GFP.The transfection of the plvx-UBE3A plasmid intoshUBE3A and HEK293 cells was carried out according to Dharmacon transfectionreagent’s protocol. After 48 h of transfection, cells weretreated with 10 μM MG132 for 4 h. Cells were harvested and centrifugedto obtain cell lysates. Cell lysates were precleared, then incubatedwith anti-substrate antibodies at 4 °C for 2 h, followed by addingprotein A/G PLUS agarose  and rockedgently overnight at 4 °C. The next day, the beads were washedfour times with cold PBS, then resuspended in 1× SDS loadingdye, and boiled. Immunoprecipitated samples were analyzed by westernblotting that was probed with an anti-UB antibody to examine the ubiquitinationlevel.The PDH enzymeactivity of HEK293 cells was measured using a colorimetric assay kit(K679) from Biovision , according to the manufacturer’sprotocol.2 incubatorto allow cells to adhere. The XF96 sensor cartridges were hydratedand placed in a 37 °C incubator, not supplemented with CO2 or oxygen/nitrogen. Before analysis, the medium of the microplatewas replaced with a new medium consisting of the Seahorse XF BaseMedium, 10 mM glucose, 2 mM sodium pyruvate, and 2 mM glutamine. Then,the microplate was placed in a CO2-free incubator for 1h before the microplate was transferred to the analyzer. Measurementswere conducted using final concentrations of 10 mM glucose, 2 μMoligomycin (OM), or 50 mM 2-deoxyglucose (2-DG).Cells wereseeded into 96-well microplates, with one well in each row or columnleft out for seeding. The empty wells were used as blank controls.The seeded plate was put into a 37 °C, 5% CO11 GC of AAVin a 50 μl volume. All animals were housed at 20–22 °Cand 50 ± 10% humidity and fed a low fat, 10 kcal % fat diet  or a high fat, 60 kcal % high-fat diet  obtainedfrom Research Diets, Inc. .All experiments were performed inaccordance with the NIH and Georgia State University guidelines forlaboratory animals’ care and use and approved by the Committeefor the Care and Use of Laboratory Animals in the Department of Biology,Georgia State University. Male C57BL/6J mice were purchased from theJackson Laboratory . AAV control viruses werepurchased from Addgene. AAV-UBE3A viruses were produced by the PennVector Core at the University of Pennsylvania. Mice were injectedinto the retro-orbital venous sinus with 103/MeOH  was used to extract lipids from theliver tissue. An aliquot of the organic phase of the extraction wascollected, dried under nitrogen, and resuspended in 1% Triton X-100.Hepatic TG and cholesterol content and serum ketone were determinedusing commercially available kits.Mice were dissected, and livers were harvested immediately and snapfrozen at −80 °C in liquid nitrogen before analysis. Bloodwas obtained by cardiac puncture and collected into EDTA-containingtubes, then centrifuged to get plasma. The liver for histology wasfixed in 10% formalin at room temperature until further analysis.Livers were homogenized in RIPA buffer  supplementedwith a 1× protease cocktail inhibitor and a 1× phosphataseinhibitor and centrifuged to obtain liver proteins. A solution ofCHClThe formalin-fixed liverwas embedded into paraffin and cut into 4 μm sections. Liversections were stained with hematoxylin and eosin (H&E).The mRNA levels of lipogenicgene expression were assessed by quantitative RT-PCR. The total RNAwas extracted from the liver samples using a Tri Reagent kit . One-step RT-PCR analysis was conductedto measure the mRNA expression using an Applied Biosystems QuantStudio3 (Thermo Fisher Scientific) with a TaqMan Universal PCR Master Mixkit . The primer and probepairs used in this analysis were purchased from Applied Biosystems(Thermo Fisher Scientific).t-test. p < 0.05 was considered statisticallysignificant.All statistical analyses wereperformed using GraphPad Prism 9.0.0 software, San Diego, CA, USA.All quantitative data were presented as mean ± SEM. Differencesbetween the two groups were assessed by unpaired Student’s xUB) with the R42E and R72E double mutations to the substrates ofa specific E3, so their identities could be revealed by proteomics.37 OUT relies on engineered interactions of the xUB–xE1, xE1–xE2, and xE2–xE3 pairs for the assembly ofthe xE1–xE2–xE3 enzymatic cascade for the exclusive delivery of xUB to the ubiquitination targets of an E3 (“x” designates the engineered UB or the transfer enzymesthat do not cross-react with their native partners in the UB transfercascade). In a previous study, we constructed the OUT cascade withUBE3A/E6AP and used it to profile the substrate specificity of UBE3Ain HEK293 cells. The OUT screen enabled us to assemble a substrateprofile of UBE3A consisting of 130 potential substrates, and amongthem, we verified multiple new substrates of UBE3A in the cells, includingkinases MAPK1, CDK1, and CDK4, protein arginine methyltransferasePRMT5, transcription factor β-catenin, UB-binding protein UbxD8,and O-linked N-acetyl glucosamine transferase (OGT).38 We further took advantage of the OUT cascade of UBE3A to compareits substrate profiles with and without the expression of HPV E6 anddiscovered that, besides p53, E6 stimulates UBE3A to ubiquitinatethe importin α family of proteins, including KPNA1–3,to induce their degradation. This would enable HPV to suppress thenuclear transport of antiviral transcription factors and pivot thehost defense against viral infection.39To profile the substrates of UBE3A, we developeda method referred to as “orthogonal ubiquitin transfer”(OUT) that would enable the transfer of a UB mutant (Supporting Information Table S1). For example,6-phosphofructokinase PFKL and PFKP, phosphoacetylglucosamine mutase(PGM3), pyruvate dehydrogenase (PDH) E1 component subunit α(PDHA1), and galactokinase (GALK1) are enzymes participating in glycolysisand the TCA cycle. Acyl-CoA dehydrogenase ACAD9 and ACADSB, very-long-chain3-oxoacyl-CoA reductase (HSD17B12), ACAT1, and acyl-coenzyme A thioesterase9 (ACAT9) are enzymes contributing to fatty acid oxidation and biogenesis.In this study, we focused on PDHA1 and ACAT1 due to their importancein glucose and lipid metabolism. PDHA1 is a key component of the pyruvatedehydrogenase complex (PDC), which catalyzes pyruvate decarboxylationto produce acetyl-CoA and CO2, hence providing the primarylink between glycolysis and the TCA cycle.42 PDHA1 downregulationhas been indicated in various types of cancer cells to enhance glycolysisactivity.43 Phosphorylation and acetylation of PDHA1could inactivate the enzyme.44 ACAT1 catalyzes thecondensation of two acetyl-CoAs to produce acetoacetyl-CoA as wellas the reverse reaction that breaks down acetoacetyl-CoA into twoacetyl-CoAs.45 In hepatic ketogenesis, ACAT1favors acetoacetyl-CoA formation, whereas, in non-hepatic ketolysis,ACAT1 functions to break down acetoacetyl-CoA.45 Due to the essential roles of ACAT1 and PDHA1 in regulatingglycolysis and lipid metabolism in the liver, we decided to verifythe ubiquitination of PDHA1 and ACAT1 by UBE3A in HEK293 cells andstudy the physiological role of UBE3A in the regulation of its substrateprotein stabilities and lipid metabolism in vivo.To decipher the role of UBE3A in regulating cell metabolism,we found several enzymes involved in sugar and lipid metabolism inthe substrate profiles of UBE3A, as revealed by the OUT screen , UbcH7(E2), and UBE3A (E3) were combined with PDHA1, and wild-type UB andATP were added to initiate the reaction. Control reactions were setup in parallel, excluding the addition of Uba1, UbcH7, or UBE3A, toverify the dependence of substrate ubiquitination on the UB transfercascades. We found UBE3A catalyzed the ubiquitination of PDHA1 inthe presence of Uba1 and UbcH7, as evidenced by the formation of thehigher molecular-weight forms of PDHA1 detected on the western blotwith an anti-PDHA1 antibody .We first performed an in vitro ubiquitination assayto verify the ubiquitination of PDHA1 by UBE3A with both the substrateprotein and the E3 ligase enzyme expressed from antibody 1A. When antibody 1A, suggeFigure S1B).37 We overexpressed UBE3A inshUBE3A cells and HEK293 cells, immunoprecipitated PDHA1, and probedits ubiquitination levels with an anti-UB antibody. We also examinedPDHA1 ubiquitination levels in shUBE3A cells and HEK293 cells withendogenous UBE3A expression. We found that PDHA1 ubiquitination waslargely undetectable in shUBE3A cells with a silenced expression ofthe E3 . These results verified thatUBE3A would recognize PDHA1 as a ubiquitination target in HEK293 cells.We then examined PDHA1 ubiquitination levels inHEK293 cells withknockdown or overexpression of UBE3A and with MG132 treatment of thecells to inhibit protein degradation by the proteasome. We screenedthe efficiency of shUBE3A plasmids in gene silencing and used theplasmid of the highest efficiency to establish stable cells with asilenced expression of UBE3A (shUBE3A) S1B.37 WeTo determine if UBE3A regulates the degradation of PDHA1 by ubiquitination,we transiently transfected UBE3A expression plasmids with titrationinto HEK293 cells and measured the steady-state levels of PDHA1 after24 h of transfection. We found that UBE3A over-expression decreasedPDHA1 protein levels in a dose-responsive manner 1C. We al47 Consistent with its role in decreasing PDHA1 protein stability,UBE3A overexpression in the cells decreased PDH activity by 20% comparedto HEK293 cells. On the other hand, there was a 20% increase in PDHactivity in shUBE3A cells compared to HEK293 cells, suggesting thatthe silenced UBE3A expression would lead to a stabilization of PDHA1and enhancement of its enzymatic activity in the cells  using the extracellular acidificationrate (ECAR) as a parameter for proton release by glycolysis. ECARvalues were expressed in units of mpH min–1, whichfollow the changes in pH in the media surrounding the cells as a resultof acidification, mainly due to glycolytic proton efflux. As expected,we observed that cells with UBE3A overexpression showed significantlyhigher basal glycolysis, glycolytic capacity, and glycolytic reservethan the mock HEK293 cells .Since PDH inactivation is associated with elevatedaerobic glucoseutilization,93 cells 2B, suggeFigure S2A). We also assessed the ubiquitinationof ACAT1 in HEK293 cells and cells with UBE3A overexpression aftertreating the cells with the proteasome inhibitor MG132 to accumulateubiquitinated species. The ubiquitination assays, which were conductedby the immunoprecipitation of ACAT1, followed by immunoblotting withthe anti-UB antibody, showed that UBE3A deficiency diminished theACAT1 ubiquitination in shUBE3A cells, which was substantially recoveredby UBE3A re-expression . Theseresults confirmed that ACAT1 is a substrate of UBE3A for ubiquitination.Following a similar assaying scheme, we verified ACAT1ubiquitination in vitro by UBE3A and found that the ubiquitinationreaction was dependent on Uba1, UbcH7, and UBE3A for transferringUB to ACAT1 3A. FurthTo determine if UBE3A regulates the stability ofACAT1 in HEK293cells, we transfected the HEK293 cells with various amounts of UBE3Aexpression plasmids. We found that UBE3A overexpression dose-dependentlydecreased ACAT1 protein levels 3C. The CFigure S3). These data suggestthat pathways other than lipogenesis might be responsible for theenhanced TG accumulation observed in the UBE3A-overexpressing liver.To determine the physiological significanceof UBE3A and its substrates in hepatic metabolism, we measured theprotein levels of UBE3A, PDHA1, and ACAT1 in the liver of mice fedwith a chow diet or HFD for 12 weeks. We observed that HFD feedingattenuated UBE3A protein levels in the liver, which was associatedwith enhanced ACAT1 protein levels compared to chow diet feeding.However, there was no difference in the liver PDHA1 protein betweenchow-fed and HFD-fed mice 4. We rea50 we examined the level of serum ketone bodies and foundthat the mice with UBE3A overexpression displayed elevated serum ketonelevels in comparison to the control mice  and free cholesterol (FC) in the livertissue. We found that mice infected with the AAV Ube3a virus exhibitedhigher TC and FC levels than the control mice infected with the AAVGFP virus 5F. Becaurol mice 5G. This 51 Excess hepatic FFAs, due to a combination of increasedFFA influx from dietary sources and adipose lipolysis, would enhancede novo lipogenesis and decrease FFA utilization via hepatic lipidoxidation and secretion and play a key role in the initiation anddevelopment of hepatic steatosis.51 Whilemuch effort has been devoted to studying the transcriptional regulationof metabolic pathways during the development of hepatic steatosis,little is known about the post-translational modification of metabolicenzymes in this process, especially through ubiquitination-mediatedmechanisms. Therefore, we employed a unique approach based on theOUT platform to identify the metabolic enzymes as ubiquitination targetsof UBE3A, which has been shown to prevent HFD-induced hepatic steatosisin mice.35 We further verified the ubiquitinationof PDHA1 and ACAT1 by UBE3A in HEK293 cells and studied the physiologicalrole of UBE3A in regulating the stabilities of its substrate proteinsand lipid metabolism in vivo. Our data indicate that the down-regulationof UBE3A by nutrient-rich diets may result in less ubiquitinationof metabolic enzymes such as ACAT1 and subsequent up-regulation ofACAT1 protein stability, thereby promoting hepatic lipid accumulation.NAFLD is a serious metabolic disorder thatstems from the dysregulationof hepatic lipid metabolism. The central feature of NAFLD is hepaticsteatosis, the deposition of excess TG in the liver.45 We confirmedthat UBE3A targets ACAT1 for ubiquitination in HEK293 cells, and theoverexpression of UBE3A accelerates the degradation of ACAT1 in thecell  that forms a heterotetramericcomplex with PDHA1 is ubiquitinated and degraded by the proteasomeupon its phosphorylation by the protein tyrosine kinase activity ofthe epidermal growth factor receptor.53 The E3 ligase responsible for the ubiquitination of PDHB is notknown. Future work is warranted to assay if UBE3A also targets PDHBfor ubiquitination and if glycolysis would be regulated by UBE3A expressionin the mouse liver.We also found that UBE3A recognizes PDHA1 as a substrateprotein,and the degradation of PDHA1 mediated by UBE3A enhances glycolysisin HEK293 cells. Since the PDH complex directs the flux of pyruvateto enter the TCA cycle and low PDHA1 activity would suppress the TCAcycle and enhance glycolysis, we identified a role of UBE3A in regulatingthe balance between glycolysis and the TCA cycle by targeting PDHA1for ubiquitination and degradation. We found that PDHA1 has similarexpression levels in the mouse liver with or without the overexpressionof UBE3A, suggesting that UBE3A-induced degradation may not play anessential role in regulating PDHA1 stability in the mouse liver 4A and 5AUBE3A gene results in the absence of the functional UBE3Aprotein in the brain, causing Angelman syndrome,55 which is a neurodevelopmental disorder characterized by delayeddevelopment, lack of speech, intellectual disability, seizure, andother symptoms.57 It was also reported that obesityis common with Angelman syndrome.59 The cohabitationof obesity with Angelman syndrome suggests that a loss-of-functionmutation in UBE3A potentially causes dysregulation of metabolic pathwaysand energy metabolism. Indeed, Ube3a+/– mice are prone to HFD-induced obesity andfatty liver development, suggesting an antagonistic role of UBE3Ain obesity-related pathogenesis.35 Mechanistically,a recent report from Kim et al. demonstrated that UBE3A suppresseslipogenic gene expression by targeting the histone methyltransferaseMLL4 for ubiquitination and degradation, resulting in the inhibitionof hepatic steatosis.35 We found that HFDfeeding down-regulates the UBE3A level in the mouse liver, suggestingthe sensitivity of UBE3A to over nutritional conditions , the first enzyme in the glycolysis pathway.61 The ubiquitination of HK-2 leads to its recognition byautophagy receptors for the degradation and inhibition of glycolysis.6-phosphofructocto-2-kinase/fructose-2,6-bisphosphotatse isoform 3(PFKB3) is another enzyme in the glycolysis pathway, and its ubiquitinationby the E3 ligase APC-Cdh1 in the neuronal cells results in the degradationof the enzyme and suppression of glycolysis. This would favor pentosephosphate pathways for glucose metabolism to generate reduced glutathione.62 In another example, CHIP E3 was found to ubiquitinatepyruvate kinase M2 (PKM2), a glycolytic enzyme and a critical regulatorof glycolysis in tumors. PKM2 ubiquitination by CHIP would destabilizePKM2 and inhibit tumor growth by suppressing the Warburg effect.63 Here, we were guided by the substrate profileof UBE3A generated by the OUT screen and authenticated ACAT1 and PDHA1as ubiquitination targets of UBE3A and established a role of the E3in regulating glycolysis and lipid metabolism. We validated OUT asan empowering discovery platform for mapping cross-regulations betweenubiquitination and metabolism pathways in the cell. We expect thatthe substrate profiles of other E3s generated by OUT, such as CHIP,E4B, and Rsp5, may also be utilized to reveal the mechanisms for maintaininga healthy cell metabolism and study how their dysregulation couldbe causative for metabolic diseases.65Thecross-regulatory mechanism between protein ubiquitination andmetabolic pathways revealed in this work may widely exist in the cellto provide checkpoints for the precise control of metabolic flux.So far, a few examples have been reported on the ubiquitination ofmetabolic enzymes to regulate their activities in the cell.In sum, we here identified PDHA1 and ACAT1as ubiquitination targetsof the E3 ligase UBE3A. Our data indicate that UBE3A-mediated ubiquitinationaccelerates the proteasomal degradation of PDHA1 and reduces theirenzymatic activities in the cells. UBE3A regulation of ACAT1 proteinstability also bears physiological significance, as overexpressingUBE3A in the mouse liver downregulates ACAT1 protein contents, increasescirculating ketone levels, and promotes hepatic steatosis. Our findingsdemonstrate a potential role for UBE3A and its target proteins inregulating hepatic nutrient metabolism.