Vibrionaceae family and is located at N-termini of proteins containing diverse antibacterial and anti-eukaryotic toxic domains. We demonstrate that RIX-containing proteins are delivered via T6SS into neighboring cells and that RIX is necessary and sufficient for T6SS-mediated secretion. In addition, RIX-containing proteins can enable the T6SS-mediated delivery of other cargo effectors by a previously undescribed mechanism. The identification of RIX-containing proteins significantly enlarges the repertoire of known T6SS effectors, especially those with anti-eukaryotic activities. Furthermore, our findings also suggest that T6SSs may play an underappreciated role in the interactions between vibrios and eukaryotes.Bacteria use the type VI secretion system (T6SS) to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition, host colonization, or when resisting predation. Identifying effectors is a challenging task, as they lack canonical secretion signals or universally conserved domains. Here, we identify a protein domain, RIX, that defines a class of polymorphic T6SS cargo effectors. RIX is widespread in the  Bacteria use the type VI secretion system (T6SS) to deliver toxic effectors into bacterial or eukaryotic cells. Here, Kanarek et al. identify a protein domain, RIX, that defines a class of polymorphic T6SS effectors with antibacterial and anti-eukaryotic toxic domains, and that enables T6SS-mediated delivery of other effectors. Nevertheless, further investigations revealed that most T6SSs play a role in interbacterial competition4, whereas only a few T6SSs have been identified as anti-eukaryotic6. These roles are mediated by toxic proteins, called effectors, which are deployed inside neighboring bacterial and eukaryotic cells8.The type VI secretion system (T6SS) is a protein delivery apparatus in Gram-negative bacteria, which was originally described as an anti-eukaryotic determinant10. The ejected apparatus is composed of Hcp proteins that are stacked as hexameric rings forming an inner tube; the tube is capped by a spike complex comprising a VgrG trimer sharpened by a PAAR repeat-containing protein (hereafter referred to as PAAR)10. T6SS effectors can be divided into two types: (1) specialized effectors, which are secreted structural components  containing a C-terminal toxic domain extension14, and (2) cargo effectors, which are toxic proteins that non-covalently interact with one of the missile components or its C-terminal extension19, either directly or aided by an adaptor protein21 or a co-effector22. Because cargo effectors can bind to diverse loading platforms on the tube or spike, they lack a canonical secretion signal or domain. Therefore, identifying cargo effectors is a challenging task, especially if they are not encoded within T6SS gene clusters or near T6SS-associated genes.Effectors are loaded onto a missile-like structure, which is ejected by a contractile sheath that engulfs it in the cytoplasm of the secreting cell23; (2) FIX (Found in type sIX) domain-containing proteins16; or (3) Rhs (Rearrangement hotspot) repeat-containing proteins28. These three domains are found N-terminal to diverse C-terminal toxic domains, and they are predicted to play a role in their delivery. MIX and FIX domains are specifically found in T6SS-secreted proteins, whereas Rhs repeats are also present in toxins secreted by other types of secretion systems. Notably, many Rhs repeats are fused to an N-terminal PAAR or PAAR-like domain and are therefore specialized effectors29. Nevertheless, many other cargo effectors lack a known domain at their N-terminus; it is possible that these effectors contain N-terminal delivery domains that have not yet been revealed.Many T6SS cargo effectors belong to one of three known classes of polymorphic toxins: (1) MIX (Marker for type sIX) domain-containing proteinsVibrio parahaemolyticus. Tme1 contains a C-terminal toxic domain named Tme (T6SS membrane-disrupting effector), which permeabilizes membranes and dissipates membrane potential30; the sequence N-terminal to the Tme domain does not contain a known domain or activity. Here, we show that the N-terminus of Tme1 is necessary and sufficient to mediate T6SS secretion in V. parahaemolyticus, and we use it to reveal a new domain that is widespread in members of the Vibrionaceae family. Proteins containing the identified domain, named RIX (aRginine-rich type sIX), are secreted via T6SS. RIX is found N-terminal to diverse C-terminal extensions with antibacterial and anti-eukaryotic toxic activities, as well as to sequences that function as loading platforms for cargo effectors. Therefore, we reveal a new class of T6SS-secreted proteins, including polymorphic effectors and secreted adaptors.We previously identified Tme1, an antibacterial T6SS effector of V. parahaemolyticus BB22OP30; its toxic domain, Tme, is located at the C-terminus. We hypothesized that the N-terminus of Tme1 plays a role in secretion via T6SS. To test this hypothesis, we monitored the expression and secretion of a truncated version of Tme1 lacking the first 60 amino acids (Tme161–310)  enabled its secretion via V. parahaemolyticus T6SS1  Fig. . As showE. coli, whereas truncating the last 85 amino acids of Tme1, corresponding to the end of the Tme domain, abrogated its toxicity. The expression of all Tme1 forms was detected in immunoblots , and Moritellaceae   of the genomes encoding RIX domain-containing proteins harbor a T6SS. Nevertheless, whereas most RIX domain-encoding strains harbor a T6SS that is similar to V. parahaemolyticus T6SS1, from which Tme1 is secreted, nearly 14% of the strains harbor a different T6SS  are fused to an N-terminal PAAR domain. These findings imply a genetic association between RIX and T6SS.Following these findings, we set out to identify regions homologous to the N-terminus of Tme1 in other proteins. We identified 502 unique protein accession numbers that contain a homologous sequence at their N-terminus (Supplementary Data 35) revealed 33 distinct clusters  antagonized this toxicity  did not lead to the killing of the prey strain. Expression of the predicted immunity protein from a plasmid in the prey strain protected it from this attack. These results indicate that the RIX domain-containing WP_005534959.1 and its downstream encoded WP_005534960.1 are an antibacterial T6SS effector and immunity pair.The results thus far indicated that: 1) Tme1 is a RIX domain-containing T6SS effector, (2) nearly all RIX domain-containing proteins are encoded by bacteria with a T6SS, and (3) most RIX domain-containing proteins have predicted C-terminal toxic domains. Altogether, these results led us to hypothesize that RIX domain-containing proteins are a new class of polymorphic T6SS effectors. To test this hypothesis, we investigated whether a predicted RIX domain-containing protein from  Tme1 is ty Fig. S. We thenein Fig. . The kilV. campbellii ATCC 25920: WP_005536620.1, WP_005530005.1, and WP_038863399.1; these proteins are predicted to have anti-eukaryotic toxic domains at their C-terminus  NaCl. Our results revealed that V. coralliilyticus BAA-450 T6SS1 is an antibacterial system that is active at 30 °C   domain  to complete a barrel-like fold , interact with a high degree of certainty  and structure prediction  analyses suggested that Rte1 contains an ADPRT (ADP ribosyl transferase)-like domain with a fold similar to that of the pertussis toxin . Notably, expression of the RIX domain-containing protein alone (pRIX) did not result in prey intoxication. Expression of Rti1 from a plasmid in the prey strain protected it from the T6SS-mediated toxicity. Taken together, our results support a role for a RIX domain-containing protein as a secreted adaptor that enables the delivery of a cargo effector via T6SS.In addition, we investigated whether Rte1 and Rti1 function as antibacterial T6SS effector and immunity pair in which the effector is dependent on the RIX domain-containing protein for delivery. To this end, we performed self-competition assays using the surrogate T6SS system. As shown in Fig. WP_095560627.1 and WP_152436132.1, these RIX domain-containing proteins are encoded by the first gene in a three-gene operon , the effector, and the immunity protein can be found fused to a PAAR domain in other vibrios. Structure predictions using AlphaFold2 suggest that the RIX-fused C-terminal domain and the N-terminus of the downstream encoded effector interact , three anti-eukaryotic effectors , and one secreted adaptor (WP_157622110.1). In addition, we identified a novel, non-RIX domain-containing T6SS cargo effector, Rte1. Notably, without RIX, the identification of these polymorphic T6SS effectors would not have been trivial, since RIX domain-encoding genes are orphan , and because many of the predicted toxic domains found in RIX domain-containing proteins have not been previously associated with T6SS effectors.In this work, we identified a new class of T6SS-secreted proteins that are widespread in V. cholerae to escape predation by grazing amoeba1, most Vibrio T6SSs investigated to date were shown to play a role in interbacterial competitions53, and only a handful of Vibrio T6SSs have been implicated in anti-eukaryotic activities. These Vibrio T6SSs include T6SS1 and T6SS3 in V. proteolyticus55, T6SS1 in V. tasmanienses56, and a T6SS in V. crassostreae57. Several additional Vibrio T6SSs were also suggested to play a role in interactions with eukaryotes based on the presence of a predicted anti-eukaryotic MIX-effector in their genome23. Furthermore, only a few T6SS effectors with anti-eukaryotic activities have been described to date, in vibrios and in other bacteria5. Therefore, our identification of diverse families of RIX domain-containing effectors with known or predicted anti-eukaryotic activities substantially enlarges this list. We hypothesize that these effectors are mostly used by vibrios to defend against predation by protists, as previously shown for V. cholerae58, rather than for colonization of aquatic or land animals. However, further work is required to identify the eukaryotic organisms that are naturally targeted by these potentially anti-eukaryotic RIX domain-containing effectors and to determine whether the role of these effectors is offensive or defensive.Revealing many RIX domain-containing proteins that have predicted anti-eukaryotic toxic domains suggests that we have underappreciated the potential role played by T6SS in the interactions between vibrios and eukaryotic organisms, whether they are hosts or predators. Although T6SS was originally described as an anti-eukaryotic determinant, allowing 15 and FIX16, are widespread across different Gram-negative bacterial families. The mechanism governing such a restricted distribution of the RIX domain is unclear. Since RIX domain-encoding genes are orphans, it is likely that they are acquired via horizontal gene transfer and not together with the T6SS apparatus. It is possible that the transfer mechanism requires a component that is found only in vibrios. Alternatively, if RIX domain-containing proteins are secreted only by T6SS types that are themselves restricted to vibrios, then the distribution of these T6SSs might provide the evolutionary drive for maintaining or discarding acquired RIX domain-encoding genes. It is intriguing to speculate that additional family-specific T6SS secretion domains are found in Vibrionaceae or in other bacterial families.Our finding that RIX domain-containing proteins are mostly restricted to vibrios reveals a previously unknown concept of family-specific T6SS secretion domains. Previously described domains that are required for T6SS secretion, such as MIX22, yet it is distinct since the RIX domain-containing protein is secreted via T6SS on its own, whereas the MIX-containing co-effector is dependent on its effector partner for secretion.In addition to effectors, RIX domains are also found in secreted T6SS adaptors, revealing a new mechanism of secreted adaptor-mediated delivery of T6SS cargo effectors. Our results indicated that RIX domains replace PAAR and VgrG proteins, which are secreted structural components of the T6SS, in modules comprising a C-terminal loading platform extension and a cargo effector and immunity pair. We experimentally demonstrated that a new cargo effector, Rte1, requires an upstream-encoded RIX domain-containing protein in order to be secreted by the T6SS and delivered into prey cells during bacterial competition. This secretion mechanism is similar to the recently described mechanism involving a MIX domain-containing co-effector59, together with our findings that RIX was able to drive the secretion of the folding-enhanced sfGFP60 and that a 526 amino acid-long RIX domain-containing protein is secreted via the T6SS. It is possible that the loading of RIX domain-containing proteins onto the ejected apparatus requires a yet-unknown component or adaptor, or that a fully assembled apparatus is required for proper effector loading. Therefore, further work is needed to determine the mechanism governing the secretion of RIX domain-containing proteins.Notably, our attempts to biochemically identify the T6SS tube-spike component with which RIX domain-containing proteins interact have been inconclusive. Therefore, the identity of the loading platform for RIX domain-containing proteins on the T6SS remains an open question. Nevertheless, we speculate that RIX-containing proteins are loaded onto the spike rather than inside the narrow Hcp tube. This hypothesis is supported by previous observations that large folded proteins are not secreted by T6SSVibrio T6SSs in interactions with eukaryotes has been underappreciated. In future work, we will determine how RIX domain-containing proteins are loaded onto the secreted T6SS, and we will explore the potential use of RIX domains and RIX domain-containing secreted adaptors to deliver engineered, non-canonical cargo effectors via T6SS to be used as bio-treatments61 or molecular biology tools62.In conclusion, we identified a new class of polymorphic T6SS cargo effectors widespread in vibrios, and we revealed a new, secreted adaptor-mediated delivery mechanism for T6SS cargo effectors. Our findings considerably enlarge the pool of predicted anti-eukaryotic T6SS effectors, suggesting that the role played by Escherichia coli strains BL21 (DE3) and DH5α (λ-pir) were grown in 2xYT broth  or on Lysogeny broth agar  plates at 37 °C, or at 30 °C when harboring effector expression plasmids. The media were supplemented with chloramphenicol (10 μg/ml) and/or kanamycin (30 μg/ml) to maintain plasmids and with 0.4% (wt/vol) glucose to repress protein expression from the arabinose-inducible promoter, Pbad. To induce expression from Pbad, L-arabinose was added to the media at 0.05-0.1% (wt/vol), as indicated.For a complete list of strains used in this study, see Table Vibrio parahaemolyticus strains BB22OP, RIMD 2210633, and their derivatives, as well as Vibrio natriegens ATCC 14048 and Vibrio coralliilyticus ATCC BAA-450, were grown in Marine Lysogeny broth  and on Marine Minimal Media (MMM) agar plates  at 30 °C. The media were supplemented with chloramphenicol (10 μg/ml) or kanamycin (250 μg/ml) to maintain plasmids. To induce expression from Pbad, L-arabinose was added to media at 0.01-0.1% (wt/vol), as indicated.Saccharomyces cerevisiae BY4741  yeast were grown in Yeast Extract–Peptone–Dextrose broth  or on YPD agar (2% wt/vol) plates at 30 °C. Yeast containing plasmids that provide prototrophy to leucine were grown in Synthetic Dropout media  supplemented with histidine (2 ml/l from a 1% wt/vol stock solution), tryptophan (2 ml/l from 1% wt/vol stock solution), uracil (10 ml/l from a 0.2% wt/vol stock solution), and glucose (4% wt/vol). For galactose-inducible expression from a plasmid, cells were grown in SD media or on SD agar plates supplemented with galactose (2% wt/vol) and raffinose (1% wt/vol).WP_015297525.1 (Tme1), sfGFP, WP_005534960.1, WP_005536620.1, WP_005530005.1, WP_038863399.1, WP_157622110.1, WP_006958655.1, and WP_050778602.1 were PCR amplified from the respective genomic DNA of the encoding bacterium or from a plasmid. Next, amplicons were inserted into the multiple cloning site (MCS) of pBADK/Myc-His, pBAD33.1F, or their derivatives using the Gibson assembly method63. Plasmids were introduced into E. coli BL21 (DE3) or DH5α (λ-pir) by electroporation, and into vibrios via conjugation. Transconjugants were selected on MMM agar plates supplemented with the appropriate antibiotics to select clones containing the desired plasmids. For galactose-inducible expression in yeast, genes were inserted into the MCS of the shuttle vector pGML10 (Riken) using the Gibson assembly method, in-frame with a C-terminal Myc-tag. Yeast transformations were performed using the lithium acetate method64.For a complete list of plasmids used in this study, see Table V. parahaemolyticus RIMD 2210633 surrogate strain and of the V. parahaemolyticus BB22OP and V. coralliilyticus BAA-450 derivatives was reported previously38. To construct deletion strains, 1 kb sequences upstream and downstream of each gene to be deleted were cloned into pDM4, a CmROriR6K suicide plasmid. The pDM4 constructs were transformed into E. coli DH5α (λ-pir) by electroporation, and then transferred into Vibrio strains via conjugation. Transconjugants were selected on agar plates supplemented with chloramphenicol, and then counter-selected on agar plates containing 15% (wt/vol) sucrose for loss of the sacB-containing plasmid. Deletions were confirmed by PCR.The construction of the E. coli strains carrying arabinose-inducible expression plasmids were grown in 2xYT broth supplemented with the appropriate antibiotics and 0.4% (wt/vol) glucose (to repress expression from the Pbad promotor) at 30 °C. Overnight cultures were washed twice with fresh 2xYT broth, normalized to an OD600 of 0.01 in 2xYT broth and then transferred to 96-well plates (200 µl per well) in quadruplicate. Cultures were grown at 37 °C in a BioTek SYNERGY H1 microplate reader with constant shaking (205 cpm). After 2 h, L-arabinose was added to a final concentration of 0.05% (wt/vol) to induce protein expression. OD600 readings were acquired every 10 min and data were visualized using GraphPad PRISM v9.For testing the toxicity of proteins during the growth of bacteria in suspension, E. coli strains carrying arabinose-inducible expression plasmids were streaked onto LB agar plates supplemented with the appropriate antibiotics and 0.4% (wt/vol) glucose (repressing plates) or 0.1% (wt/vol) L-arabinose (inducing plates). Plates were incubated for 16 h at 37 °C.For testing the toxicity of proteins during the growth of bacteria on solid media, E. coli cultures harboring arabinose-inducible expression plasmids were grown overnight in 2xYT broth supplemented with appropriate antibiotics to maintain the expression plasmids. Bacterial cultures were then washed with fresh 2xYT and resuspended in 3 ml of 2xYT supplemented with appropriate antibiotics. Next, bacterial cultures were incubated with constant shaking (220 rpm) at 37 °C for 2 h. After 2 h, L-arabinose was added to a final concentration of 0.05% (wt/vol) to induce protein expression, and cultures were grown for 2 additional hours. Cells equivalent to 0.5 OD600 units were collected, and cell pellets were resuspended in 50 µl of 2x Tris-glycine SDS sample buffer . Next, samples were boiled at 95 °C for 10 min, and then loaded onto TGX stain-free gels (Bio-Rad) for SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, which were immunoblotted with α-FLAG , α-Myc , or custom-made α-VgrG165 antibodies at 1:1000 dilution, as indicated. Finally, protein signals were visualized in a Fusion FX6 imaging system (Vilber Lourmat) using enhanced chemiluminescence (ECL).64 supplemented with 4% (wt/vol) glucose, washed twice with ultrapure milli-Q water, and then normalized to an OD600 of 1.0 in milli-Q water. Next, 10-fold serial dilutions were spotted onto SD agar plates containing either 4% (wt/vol) glucose (repressing plates) or 2% (wt/vol) galactose and 1% (wt/vol) raffinose (inducing plates). The plates were incubated at 30 °C for two days.Yeast cells were grown overnight in appropriate SD media600 = 1.0 in selective media supplemented with galactose and raffinose to induce protein expression. Next, the cells were grown at 30 °C for 16 h, and 1.0 OD600 units of cells were pelleted and lysed, as previously described64. Proteins were detected in immunoblots using α-Myc antibodies at a 1:1000 dilution and α-β-actin antibodies  at a 1:500 dilution.Yeast cells harboring galactose-inducible expression plasmids were grown overnight in selective SD media supplemented with glucose, washed twice with ultrapure milli-Q water, and then normalized to ODVibrio strains were grown overnight, normalized to an OD600 of 0.5, and then mixed at a 4∶1 (attacker:prey) ratio in triplicate. Then, 25 μl of the mixtures were spotted onto MLB agar competition plates supplemented with 0.05% (wt/vol) L-arabinose when induction of protein expression from a plasmid was required. Competition plates were incubated at the indicated temperatures for 4 h. The colony-forming units (CFU) of the prey strains at t = 0 h were determined by plating 10-fold serial dilutions on selective media plates. After 4 h of co-incubation of the attacker and prey mixtures on the competition plates, the bacteria were harvested and the CFUs of the surviving prey strains were determined by plating 10-fold serial dilutions on selective media plates. Data were visualized using GraphPad PRISM v9. Statistical tests were performed in Excel 2019.Attacker and prey Vibrio strains were grown overnight in appropriate media and then normalized to an OD600 of 0.18 in 3 ml MLB broth supplemented with appropriate antibiotics and 0.01-0.05% (wt/vol) L-arabinose, when expression from an arabinose-inducible plasmid was required. Bacterial cultures were incubated with constant shaking (220 rpm) at the indicated temperatures for the specified durations. For expression fractions (cells), cells equivalent to 0.5 OD600 units were collected, and cell pellets were resuspended in 50 µl of 2x Tris-glycine SDS sample buffer . For secretion fractions (media), supernatant volumes equivalent to 10 OD600 units were filtered (0.22 µm), and proteins were precipitated using the deoxycholate and trichloroacetic acid method66. The precipitated proteins were washed twice with cold acetone, and then air-dried before resuspension in 20 µl of 100 mM Tris-Cl (pH = 8.0) and 20 µl of 2x protein sample buffer with 5% β-mercaptoethanol. Next, samples were incubated at 95 °C for 5 or 10 min and then resolved on TGX Stain-free gel (Bio-Rad). The proteins were transferred onto 0.2 µm nitrocellulose membranes using Trans-Blot Turbo Transfer (Bio-Rad) according to the manufacturer’s protocol. Membranes were then immunoblotted with α-FLAG , α-Myc , Direct-BlotTM HRP anti-E. coli RNA polymerase sigma 70  or custom-made α-VgrG165 antibodies at 1:1000 dilution. Protein signals were visualized in a Fusion FX6 imaging system (Vilber Lourmat) using ECL reagents.V. parahaemolyticus BB22OP. Five iterations of PSI-BLAST were performed against the RefSeq protein database. In each iteration, a maximum of 500 hits with an expect value threshold of 10−6 were used. Compositional adjustments and filters were turned off. The genomic neighborhoods of RIX domain-containing proteins  of RIX was constructed using the N-terminal 55 residues of Tme1 WP_015297525.1) from 97525.1 f67. Aligned columns not found in the RIX domain of Tme1 were discarded. The RIX domain-conserved residues were illustrated using the WebLogo server68 (https://weblogo.threeplusone.com).RIX domain sequences were aligned using Clustal OmegarpoB were aligned using MAFFT v7.505 FFT-NS-2 69. Partial and pseudogene sequences were discarded. The evolutionary history was inferred using the neighbor-joining method70 with the Jukes-Cantor substitution model (JC69). The analysis included 686 nucleotide sequences and 4,021 conserved sites. The tree was visualized using iTOL (https://itol.embl.de/).DNA sequences of 71 in August 2022 . The structures of proteins that were not available on the database and of protein complexes were predicted in ColabFold: AlphaFold2 using MMseqs236. All PDB files used in this work are available as Supplementary Data 72.Predicted protein structures were downloaded from the AlphaFold Protein Structure Database35. To predict the activities or domains in each cluster, at least two representative sequences (when more than one was available) were analyzed using the NCBI Conserved Domain Database (CDD)73 and HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred)43. If no activity or domain could be predicted, the protein sequences were further used for AlphaFold2 structure prediction36 followed by a 3D protein structure comparison in the Dali server44. Predicted antibacterial or anti-eukaryotic activities were determined based on known targets of the C-terminal toxic domains  and by the presence or absence of possible immunity gene immediately downstream of the RIX domain-encoding gene . Notably, for some RIX domain-containing proteins, the activity was experimentally confirmed in this or in past work .Amino acid sequences C-terminal to RIX were clustered in two dimensions using CLANSVibrio coralliilyticus were identified using PSI-BLAST . Rte1 homologs were aligned using Clustal Omega and conserved residues were illustrated using WebLogo 3 (https://weblogo.threeplusone.com/create.cgi).Homologs of Rte1 (WP_006958655.1) from Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data S1Supplementary Data S2Supplementary Data S3Reporting SummarySource Data


In recent years, there has been a growing demand for gluten-free and functional products, driven by consumer preferences for healthier and more diverse food choices. Therefore, there is a need to explore new ingredients that can be used as alternatives to traditional gluten-containing grains. Thus, this work evaluated the physical, chemical, technological, and sensorial properties of extrudates and cookies from composite tannin sorghum (rich in resistant starch) and white cowpea flours. Extrudates and cookies were produced from a composite flour made of sorghum and cowpea, at a sorghum:cowpea flour ratio of 70:30, 50:50, and 30:70. Then, raw flours, cookies, and extrudates were characterized . Results obtained for particle size distribution and bulk density indicated that the particles increased and the color changed with the addition of cowpea flour. The raw tannin sorghum flour had a higher resistant starch concentration (36.3%) and antioxidant capacity (211.2 µmolTE/g), whereas cowpea flour had higher levels of proteins (18.7%) and dietary fiber (20.1%). This difference in the raw flour composition contributed to the nutritional value of the extrudates and cookies, especially the cookies which undergo dry heat and had higher retention of resistant starch and antioxidants. Moreover, sorghum flour presented a higher tendency to retrograde (high setback), which was decreased by the addition of cowpea flour. Overall acceptance and intention to purchase were higher for extrudates with 100% sorghum flour  and cookies with 70% cowpea flour . Therefore, nutritious and functional gluten-free extrudates and cookies, of good acceptability, can be produced from composite tannin sorghum and white cowpea flours. According to a report by Grand View Research, the size of the global gluten-free product market was valued at USD 5.9 billion in 2021 and is expected to grow at a compound annual growth rate (CAGR) of 9.8% from 2022 to 2030. This growth can be attributed to an increasing prevalence of celiac disease, rising consumer awareness regarding gluten intolerance, and the availability of a wide range of gluten-free products in the market [Sorghum bicolor) is a versatile, drought-tolerant cereal commonly grown in semi-arid regions of Africa, Asia, Australia, and North and South America [Sorghum  is a nutritious crop that thrives in locations unsuitable for growing most other edible legumes as it is heat- and drought-tolerant, making it an environmentally and climate-friendly crop [Phaseolus vulgaris) and chickpea (Cicer arietinum) [Cowpea (dly crop . It is aietinum) ,10. Alonietinum) ,12,13.In addition to the numerous health benefits associated with the consumption of the bioactive compounds present in sorghum and cowpea separately, researchers have also reported that the synergistic interaction of bioactive compounds from these grains can enhance their bioavailability, leading to improved health benefits ,15. For Despite possessing many bioactive compounds and almost similar nutritional content compared to other cereals and legumes, products from sorghum and cowpea on both small and large scales are far below their potential . MoreoveFor instance, cowpea has been used to improve the technological and nutritional profiles of baked and fried products, comminuted meat products like chicken nuggets and meatballs, and soups, stews, and salads ,21,22,23A sorghum genotype with high tannin and resistant starch content (BRS 305) was obtained from Embrapa Maize and Sorghum . A commercial white cowpea (“Feijão Fradinho”) was kindly donated by Granfino Alimentos . Ingredients used to make the cookies , egg, cocoa powder, and Royal baking powder) were purchased from Escola supermarket at UFV . Hydrochloric acid and sulfuric acid were purchased from Synth, sodium carbonate from Alphatec, and petroleum ether from Quimica. Folin–Ciocalteau, sodium carbonate, Trolox, ABTS, vanillin, maleic acid, gallic acid, and ethanolamine were purchased from Sigma Aldrich. The dietary fiber assay kit and resistant starch assay kit were purchased from Megazyme Ltd., Wicklow, Ireland.w/w) in a homogenizer Y shape TE-201/05  for 15 min to homogenize the flour particles. In addition, 100% sorghum and cowpea flours served as controls, totaling 5 samples: 100S, 70S:30C, 50S:50C, 30S:70C, and 100C. Flour particle size distribution was measured in water using a Mastersizer S3500 particle size analyzer . Values of D (10) (83 µm), D (50) (330 µm), and D (90) (1600 µm), which represent the maximum particle diameter below which 10%, 50%, and 90% of the sample fall, respectively, were obtained. All the measurements were carried out in two replications. The produced composite flours were dried in an oven with air circulation at 35 °C for 15 h, cooled, and stored in polyethylene packages at a temperature of −22 °C until needed for analysis.Matured dried seeds of sorghum and cowpea were sorted to remove damaged seeds, foreign materials, dirt, and stones. The grains were ground in a laboratory hammer mill model 3100  with a 0.8 mm opening. In order to produce the composite whole grain flours, sorghum and cowpea flours were mixed at ratios (sorghum:cowpea) of 70:30, 50:50, and 30:70  were evaluated according to the CIELAB system, in a colorimeter CR-400 , using illuminant D65 and observer angle 2°, and the readings were performed using six replicates per sample.The chemical composition of the raw flours, extrudates, and cookies was analyzed according to the AACC  officialv/v) (1:10 w/v) by stirring for 1 h at room temperature (25 °C) on a magnetic stirrer. The suspension was centrifuged at 3100× g for 10 min. After this time, the supernatant was removed and used for the determination of free phenolics. The method used was the Folin–Ciocalteau method as described by Blainski et al. [The extraction of free phenolic compounds from samples was carried out using ethanol as the solvent. The samples were mixed with ethanol in water  of the samples was determined according to the ABTS  method. The resistant starch (RS) content of the samples was measured according to the resistant starch assay kit from Megazyme International  (AACC method 32-40). Total starch (TS) was measured using the Total starch assay kit from Megazyme (AACC method 76-13).A Rapid Visco Analyzer series 4 RVA  was used to measure the paste viscosities of the raw flours, extrudates, and cookies according to the methodology reported by Carvalho et al. . The pasThe texture of the cookies was determined using the TA-XT Plus texture analyzer  using a 50 kg load cell, equipped with a three-point bend rig (HDP/3PB) and a heavy-duty platform. Maximum force was recorded as the hardness value and test conditions were pre-test speed of 1 mm/s, test speed of 3 mm/s, post-test speed of 10 mm/s, and penetration distance of 5 mm. The readings were performed on 10 cookie samples.The texture of extrudates was measured using the method described by Alzuwaid et al.  with sliApproval was sought before recruiting participants. The approval number was CAAE: 63482522.3.0000.5268. Sensory evaluation of extrudates and cookies was done separately, using 60 panelists for each product. Participants who identified as frequent consumers of breakfast cereals and cookies were recruited from staff and students at Universidade Federal De Viçosa, via posters and WhatsApp messages. Participants were provided with an information sheet and consent forms. People with any food allergies were excluded from the evaluation.Three samples of extrudates  and five cookie samples  were subjected to evaluation by the participants using a 9-point Hedonic scale as described by Larmond  with scop < 0.05 was considered as significant. SPSS Statistics v.22  was used for all analyses.All data were reported as mean ± standard deviation. One-way ANOVA with Tukey post-hoc test was used to identify significant differences between samples for all measured parameters. The average particle size of the flours used for the production of the samples (extrudates and cookies) is shown in 3 (The bulk density obtained for the flours in this study increased with the addition of cowpea flour and ranged between 0.09–0.17 g/cm3 . The comFlour with low bulk density occupies more volume for a given mass compared to flour with higher bulk density . In foodp > 0.05) and ranged from 7.48–9.71%. Cookies produced from 50% sorghum and 50% cowpea flour had the lowest value (7.48) for weight loss, while cookies from 30% sorghum and 70% cowpea flour had the highest value (9.71). Weight loss results from the evaporation of water in dough during baking. The addition of cowpea flour to cookies did not play a significant role in weight loss.The physical properties of cookies  and extrudates (color) are shown in p < 0.05) and ranged from 5.18–5.45, with sample 30S:70C having the highest value and sample 100S the lowest value. The addition of cowpea flour increased the expansion slightly. This increase could be attributed to the fine particle size of the cowpea flour. Cowpea produced a large amount of fine dust during milling and had a particle size of less than 500 µm. This theory is backed by the findings of Moraru and Kokini [The expansion ratio of cookies differed significantly , redness (a*), yellowness (b*), chroma (C), and hue (H) of extrudates and cookies are shown in All the observed color modifications were related to the natural color of the added ingredients but could also have been from the brown pigments generated due to the Maillard reaction .The proximate composition of whole grain flours, extrudates, and cookies is shown in p > 0.05). This could be because the fat and ingredients used in the cookie’s formulation were standardized for all samples, and their interaction could have increased the stability of the fats. However, the fat content obtained in this study was within the range of fat content for commercial cookies (20–70%), indicating a healthier cookie.The fat content ranged between 0.84–2.95, 0.34–0.44, and 22.11–24.20% for flours, extrudates, and cookies, respectively. The inclusion of cowpea flour slightly affected the fat content of the extrudates produced from the composite flour. This finding suggests that cowpea flour can be added to cereal extrudates without changing the foods’ nutritional fat content. When compared to raw flour, extrusion reduced the fat content of the extrudates . De Pillp < 0.05) higher protein content than the sorghum control sample (100% Sorghum)  . The incp < 0.05) the ash content of the samples  and ranged between 0.90 to 45.34, 0.62 to 2.56, and 1.48 to 7.16 mg GAE/g for flours, extrudates, and cookies, respectively. The free phenolic compounds present in sorghum flour were higher than that in cowpea flour. Generally, the same trend was observed both in the extrudates and cookies as the amount of phenolics decreased with an increase in the percentage substitution of cowpea flour. The data obtained for the phenolic content of extrudates agreed with that of de Moraes et al. [The results for the free phenolics, tannin, antioxidant capacity, and resistant starch contents of the flours, extrudates, and cookies are shown in s et al. , who asss et al. , noted tFurthermore, the results obtained for cookies agreed with that of Chiremba et al. , who repp < 0.05). The values obtained ranged between 0.65 to 47.25, 0.38 to 5.53, and 2.30 to 30.51 mg Catechin equiv./g. The reduction followed the same pattern for the extrudates and cookies, as it was proportional to the percentage increase in the substitution of cowpea flour. However, the cookies retained more of their tannins compared to the extrudates. This could be related to the different heat treatments applied in processing the products, as the high temperature and pressure used during extrusion have been reported to lead to more degradation of tannins compared to baking [The result for the tannin content of flour, extrudates, and cookies as shown in o baking . The redo baking , or the o baking  and otheo baking . Dlaminio baking  added tho baking  and Dlamo baking  reportedo baking  reportedp < 0.05) and ranged from 20.10–211.20, 7.40–38.10, and 12.60–60.20 Micromole TE/g sample for flour, extrudates, and cookies, respectively. The sorghum flour had the highest value, which could be linked to the high content of phenolics and tannins present in the grain as seen in About the antioxidant capacity, the values obtained for the samples differed significantly  tend to retain higher resistant starch content from the original flour used, compared to wet heat processing (extrudates) . In addi−1 reported by da Silva et al. [The values for textural properties of extrudates obtained via the puncture test are summarized in a et al.  for extra et al. ,39.The average specific force of structural ruptures (Fsr) was also low, with values ranging from 0.02–0.04 N. The values were lower than the 0.30–1.09 N reported by da Silva et al.  for extrIn addition, the results for crispness work (Wc) ranged between 0.31 and 1.98 N mm . This prp < 0.05) and ranged between 9.06–21.02 N. The cookies from the 70S:30C blend had the highest value for hardness. This could be a result of the higher bulk density recorded for this blend and the lower moisture content of the sample. Interestingly, the results obtained in this study were similar to that of Garźon et al. [The results of the texture of extrudates are presented in n et al. , who repn et al.  reportedGenerally, the results did not follow a trend, which could be because of the large variability in the particle size of the flour. Belorio et al.  noted thp < 0.05). A remarkable reduction was observed in the pasting properties of the cookies and extrudates when compared to that of raw flours. Generally, the pasting properties were seen to dwindle with the substitution of cowpea flour.The results of the pasting properties of flour, extrudates, and cookies are shown in p < 0.05) and ranged between 26.50 to 49, 52 to 87, and 33.50 to 48.00 cP for flour, extrudates, and cookies, respectively. Trough viscosity measures the ability of paste to resist breakdown during cooling. The results obtained for trough in this study indicate that raw sorghum and cowpea starch granules had better resistance against breakage compared to their processed counterparts. The result agreed with the findings of Hashimoto et al. [p < 0.05) than BRS Novaera. Similar results were also obtained by Wang et al. [The trough viscosities obtained In this study differed significantly  from the rest of the flour and cookie samples, while the extrudate made with 70S:30C extrudate had the highest value for peak viscosity. The peak viscosities of the flour and extrudates decreased with an increase in the substitution of cowpea flour; those of the cookies did not follow any trend. The low peak viscosities exhibited by the extrudates and cookies could be an indication of various levels of starch depolymerization and gelatinization that occurred during extrusion and baking [In addition, the peak viscosities of samples ranged from 766.50 to 1069, 53 to 123, and 75 to 95.50 cP for flour, extrudates, and cookies, respectively. The 100% sorghum flour and 100% sorghum cookies exhibited the highest peak viscosities and were significantly different  from the rest of the samples. The breakdown viscosity is an index of the stability of the starch and a measure of the ease with which the swollen granules can be disintegrated [Furthermore, the values obtained for breakdown viscosities ranged from 730.50 to 1042.50, 41.00 to 82.00, and 38.50 to 57.50 cP for flour, extrudates, and cookies, respectively. The 100% sorghum flour had the highest breakdown and was significantly different  from other flour samples, while extrudates from 30S:70C had the least value among the extrudates. The low setback values observed in the extrudates and cookies after the addition of cowpea flour also indicated a reduction in starch retrogradation, which could be related to the depolymerization of the starch and its subsequent complexation with other components in sorghum and cowpea flours [In addition, the setback viscosities ranged from 736.00 to 1639.00, 1.00 to 28.00, and 22.00 to 70.00 cP for flour, extrudates, and cookies, respectively. The 100% sorghum flour had the highest values for setback and was significantly different  in the sensory attributes among the extrudate samples, except for aroma. The sensory attributes were seen to decrease as the percentage increased in cowpea flour addition. The extrudate made from 100% sorghum flour had the highest value for all sensory parameters evaluated, and had an intention to purchase score of 68.33%, indicating an acceptance of the product. According to Awika and Rooney [The sensory attributes of extrudates and cookies from composite flours of sorghum and cowpea are shown in d Rooney , the senp > 0.05) amongst the samples. Sample 30S:70C had the highest score for all sensory parameters measured, except for appearance. Sample 100S had the highest value for appearance. Samples 30S:70C and 70S:30C had the same value for intention to purchase (76.67%). The high acceptance of the sample could be a result of the nutty flavor developed in cookies substituted with cowpea flour during baking, which most of the panelists considered acceptable. Similarly, earlier research found that cookies manufactured with gluten-free flour had sensory scores that were higher than 5 for all sensory qualities [On the other hand, the sensory scores of the cookies did not differ significantly  were the best samples, as they were observed to retain more of the chemical properties of their raw flour and had good acceptability. Sorghum flour can be substituted with cowpea flour, up to 50% for extrudates and 70% for cookies, without affecting the sensorial properties of the final products. These findings suggest that composite sorghum and cowpea flours can be used to develop nutritious gluten-free and functional foods that are acceptable to consumers. Further research is needed to optimize the formulations and processing conditions to enhance the properties of the final products.


Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the N-terminus of the HTT gene. The CAG repeat expansion translates into a polyglutamine expansion in the mutant HTT (mHTT) protein, resulting in intracellular aggregation and neurotoxicity. Lowering the mHTT protein by reducing synthesis or improving degradation would delay or prevent the onset of HD, and the ubiquitin-proteasome system (UPS) could be an important pathway to clear the mHTT proteins prior to aggregation. The UPS is not impaired in HD, and proteasomes can degrade mHTT entirely when HTT is targeted for degradation. However, the mHTT protein is differently ubiquitinated when compared to wild-type HTT (wtHTT), suggesting that the polyQ expansion affects interaction with (de) ubiquitinating enzymes and subsequent targeting for degradation. The soluble mHTT protein is associated with several ubiquitin-modifying enzymes, and various ubiquitin-modifying enzymes have been identified that are linked to Huntington’s disease, either by improving mHTT turnover or affecting overall homeostasis. Here we describe their potential mechanism of action toward improved mHTT targeting towards the proteostasis machinery. HTT) gene  is an inherited neurodegenerative disease caused by a CAG repeat expansion in exon1 of the Huntingtin (TT) gene . The CAGTT) gene , as obseTT) gene . The symTT) gene . AlthougTT) gene . The autTT) gene . The proe itself . In addie itself  and ubiqe itself  are not e itself , with diUbiquitin is a highly conserved small (8.5 kDa) modular signaling protein that functions as a post-translational modification and can be used to target proteins for degradation. Ubiquitin is attached mainly to internal lysine residues in target proteins . Additioen bloc, DUBs that cleave within chains, and DUBs that remove single ubiquitin molecules from the tip of the polyubiquitin chain  proteins. UBA1 and UBA6 can activate ubiquitin, and contain an N-terminal adenylation domain to bind ATP and ubiquitin, a catalytic cysteine domain, and a C-terminal ubiquitin-fold domain for E2 recruitment. E1 enzymes function on the apex of the ubiquitination pathway to activate ubiquitin with the use of ATP and transfer it to the E2 enzyme . UBA1 sei.e., the Ubiquitin Conjugation (UBC) domain  domain , which f) domain . Class I) domain . The dife detail .E3 ubiquitin ligases are enzymes that recognize substrates that need to be ubiquitinated. Based on differences in structure, E3 ubiquitin ligases can be divided into four types: HECT type, RING-finger type, U-box type, and RBR type .via their N-termini and transfers ubiquitin to the substrates. In contrast, RING-finger E3 ligases do not receive the ubiquitin molecule from the E2 enzyme. RING-finger E3 ligases are described below.The human HECT E3 ligase family consists of 28 members, which all vary in their N-terminal domain, used for substrate recognition , while tbona fide catalytic center. Instead, the ubiquitin molecule is transferred directly from the E2 onto the substrate. RING-finger E3 ubiquitin ligases thus function as scaffolds between the E2 and the substrate. Many variations exist between RING-finger E3s as they could function as monomeric proteins but could also be part of large multi-subunit complexes, such as cullin-RING ligases and the anaphase-promoting complex/cyclosome (APC/C) complex. Also, the substrates of RING-finger E3s are highly varied, as they might recognize multiple different substrates, and various E3 ubiquitin ligases could also recognize a specific substrate. The ligase activity and substrate recognition can be modulated via post-translational modifications. RING-type E3 ligases have been reviewed in more detail elsewhere -finger E3 ubiquitin ligases comprise, with over 600 members, the largest family of E3 ubiquitin ligases. RING-finger E3s share a RING domain, which is used to interact with E2 enzymes, but this domain does not interact with ubiquitin as it lacks a lsewhere .B. rapa  E3s, and this family has 14 members in humans. RBR ligases contain a RING1 domain, followed by a central in-between-RINGs (IBR) domain and a RING2 domain. The RING1 domain recruits the ubiquitin-loaded E2 and transfers ubiquitin to the catalytic cysteine in the RING2 domain, which transfers it to the substrate . Since RThe ubiquitination of protein substrates like HTT becomes reversible through the action of deubiquitinating enzymes (DUBs), which can cleave off ubiquitin in two ways. They can either recognize and target specific protein interaction domains or show specificity for particular polyubiquitin linkage types. The latter type of DUB may hydrolyze polyubiquitin chains but leave the proximal ubiquitin on the substrate. Linkage specificity is determined by the catalytic domain, ubiquitin-binding domains, or by the specificity of interacting proteins. Apart from linkage selectivity, DUBS could also show selectivity in removing off ubiquitin moieties from the end of polyubiquitin chains or cleaving within chains. There are seven different families of DUBs. Six of them are classified as cysteine proteases: USPs, UCHs, OTUs, MJDs , MINDYs, and ZUP1. The seventh family is the JAMM family, also known as MPN, and is classified as metalloproteinase. DUB specificity and regulation have been reviewed elsewhere in more detail .Aberrant splicing and protein cleavage can generate different mHTT protein fragments, including the N-terminal exon1 fragment containing the polyQ expansion. These fragments were shown to be highly pathogenic in HD mouse models and are also observed in fibrillar aggregates in brains of HD patients . The mHTSeveral ubiquitin ligases and DUBs have been linked to HD. These enzymes might be potential therapeutic targets to facilitate the degradation of mHTT by the proteostasis machinery prior to aggregation. Here we give some background information about the enzymes and describe how these enzymes were linked to HD. For instance, the enzymes might affect proteasome-dependent degradation of mHTT, or affect solubilization, polymerization, or aggregation of mHTT. Eventually, the ubiquitin-modifying enzymes could affect cellular pathways and cellular stress that affect HD pathogenicity directly or indirectly. We also describe whether the ubiquitin-modifying enzymes might be a potential target for HD therapy. We start with discussing the involved E1 enzyme, followed by E2 enzymes, and finally E3 enzymes, which are categorized by type, starting with HECT E3s, followed by RING-finger E3s, RBR E3, and finally U-box E3s. The DUBs are described per class and USP type of DUBs in alphabetical order. The effects of these ubiquitin-modifying enzymes on mHTT levels, interactions, aggregation and pathology are summarized in via the UPS. Nollen and colleagues performed a genome-wide RNA interference screen in C. elegans and identified UBA1 as one of the genes that increased polyQ-peptide aggregation upon knockdown  is an E1 ubiquitin-activating enzyme and catalyzes the first step of the enzymatic ubiquitination cascade; namely activation of the ubiquitin molecules and subsequent transfer to E2 enzymes. Therefore, UBA1 plays a key role in the regulation of ubiquitin homeostasis, hence ubiquitination is involved in all downstream cellular processes, including protein degradation nockdown . Wade annockdown . Despitenockdown . While Uvia the formation of Lys63/Lys48-linked polyubiquitin signals  belongs to the class II E2 enzymes, which contain a Ubiquitin-Associated (UBA) domain C-terminal to the UBC domain. A UBA domain binds to ubiquitin, polyubiquitin chains, and ubiquitinated proteins. HIP2 can elongate K48-linked polyubiquitin chains and targets substrates for degradation , yet the signals . HIP2 inng assay . Colocalng assay . While kng assay , suggestng assay . Since Hng assay , this suns model . The disvia K48-linked polyubiquitin. Since the HTT N-terminus is also largely unstructured and disordered , also known as E6AP, is a HECT domain E3 ubiquitin ligase with a zinc-binding fold called the AZUL  domain. UBE3A assembles K48-linked polyubiquitin chains  and targ repeats . In addi repeats . While t repeats . In cont2A cells . Since t2A cells . Overexp2A cells  and promtination , and in tination . Knockdotination  and redutination . In additination . Taken tvia polyubiquitination  is a multifunctional HECT domain E3 ubiquitin-ligase and belongs to the NEDD4 subfamily. WWP1 contains an N-terminal C2 domain for membrane and protein binding, four tandem WW binding domains for substrate binding, and a C-terminal catalytic HECT domain for ubiquitin-protein ligase activity for both mono- and polyubiquitination . WWP1 prHACE1 mRNA in a mouse striatal cell line. HACE1 overexpression made mHTT-expressing striatal cells less sensitive to oxidative stress. This effect was independent of HACE1s E3 ligase activity but rather due to NRF2 induction (HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) is a HECT-type E3 that contains six Ankyrin repeats at the N-terminus and a C-terminal HECT domain. Ankyrin repeats are relatively common protein domains of ∼33 amino acids in length. These repeats act as scaffolds to facilitate specific protein-protein interactions through variable surface exposure . HACE1 induction . NRF2 wanduction . YAC128 nduction . However. The TRAF domain consists of two distinct subdomains: the TRAF N-domain, which is a coiled-coil domain, and a highly conserved TRAF-C domain  is a RING E3 ligase and member of the TRAF protein family and contains an N-terminal RING domain, four zinc finger (ZF) motifs, and a C-terminal TRAF domain. The N-terminal RING and ZF1 domains are used for K63 polyubiquitination . The TRAC domain . The TRAC domain . TRAF6 pC domain , and is C domain , K48/K63C domain , as well disease . TRAF6 ipatients . Overexppatients . DespiteDrosophila SCA3 model, another polyQ aggregation disorder. To conclude, there seems to be a link between PJA1 and HTT, irrespective of the polyQ repeat length. However, the exact role in HD and the affected mechanism remains to be resolved.PJA1 or PRAJA1/RNF70 is a RING ubiquitin ligase with yet unknown function. It is expressed in various tissues with the highest expression levels found in brain and has been associated with neurodevelopmental disorders, such as epilepsy and/or craniofacial abnormalities . The expRecently it was shown that cytoplasmic and nuclear aggregates of mHTT have a different biochemical composition, as well as different interactome and structural properties, which suggests that different mechanisms drive aggregate formation, clearance, and toxicity, depending on the subcellular localization . When thvia ubiquitination. Auto-ubiquitination of the HRD1 RING domain generates a high-affinity substrate binding site on the cytoplasmic side of HRD1, where it ubiquitinates these substrates  and mediates the degradation of misfolded proteins in ER-associated protein degradation (ERAD). In ERAD, HRD1 functions as a pore and can regulate the transport of misfolded proteins from the ER lumen to the cytoplasm bstrates . Next, pbstrates , suggestbstrates . Althougbstrates . HRD1 imvia the ERAD pathway  is a RING-BETWEEN-RING (RBR) E3 ligase that is located in the cytoplasm. It contains an N-terminal UBL domain and four zinc-coordinating RING-like domains: RING0, RING1, IBR, and RING2. The UBL domain controls cellular PRKN levels and activity in addition to binding to SH3-, ubiquitin interacting motif (IUM) domains and the proteasome complex . Polyubi pathway . It inte pathway . The E3  pathway . Activat the UPS . Increas the UPS , and PRK the UPS . When thobserved . This coobserved .via the proteasome. Since linear ubiquitination is a very specific process, it might be a promising target for drug development for HD and other protein aggregation-based diseases (The E3 ubiquitin ligase HOIP belongs to the RBR family and is the only known E3 ligase that can assemble linear M1-linked polyubiquitin chains and functions as part of the LUBAC complex . HOIP usdiseases .via three tetratrico peptide repeat (TPR) domains, and interacts with the proteasome via its E4/U-box domain. Thus, CHIP possibly acts as a regulator in triage decisions; it sends the substrate for refolding or proteasome-dependent degradation. CHIP uses its TRP domain, not the E4/U-box domain, to reduce aggregation of polyQ-GFP and GFP-tagged N-terminal mHTT in transfected cell lines, suggesting that polyQ proteins are refolded through the action of CHIP in order to keep them in a soluble state and hence reduce aggregation (Drosophila (Drosophila model for HD (The C-terminus of HSP70-interacting protein (CHIP) is a U-box ubiquitin ligase (UULs) that contains a U-box domain structurally related to the RING finger domain. CHIP is an E3 ubiquitin ligase that links the protein folding machinery with the UPS, interacts with molecular chaperones HSP70, HSC70, and HSP90 regation . CHIP leregation . Interesregation . Colocalregation . Studiesregation . In addiosophila . In lineosophila . While oosophila . In addil for HD . Their cl for HD . In addil for HD . The autDrosophila model of HD. Expression of a dominant negative mutant of CUL1 resulted in increased aggregation in cell culture. The silencing of CUL1 or SKP1 in Drosophila resulted in increased aggregation and toxicity. Since the combination of mHTT expression and CUL1 or SKP1 silencing was lethal, results were based on the few surviving animals  is a multiprotein E3 ubiquitin ligase complex that belongs to the cullin family of RING-finger E3s. The SCF complex consists of three core subunits , and a variable F-box protein. Independent of the SCF complex, the F-box protein recognizes and interacts with specific target proteins. Substrate recognition often depends on post-translational modifications, and each F-box protein may recognize several different substrates. Next, the F-box protein binds to the adaptor protein SKP1 that links the F-box protein to CUL1, thereby recruiting the substrate to the E2 enzyme and the RBX1 domain. RBX1 contains a RING-finger domain to which the E2 enzyme binds. Different Cullins and F-Box Proteins combinations can generate a wide variety of E3 ubiquitin ligases targeting different substrates. The SCF complex targets proteins with mono- or polyubiquitin chains and can regulate various molecular processes, including protein localization, protein activity, or protein targeting for degradation, of which the latter one is best studied. In this case, the SCF complex is involved in the polyubiquitination of target proteins. Bhutani and colleagues found that the levels of CUL1 and SKP1 were reduced in HD mice brain. In addition, lower CUL1 levels were observed in a cellular model and a  animals . Gomez-P animals . The proAtaxin 3 (ATXN3) belongs to the Josephin family of deubiquitinating enzymes and contains an N-terminal Josephin domain (JD) with a papain-like fold, while the C-terminus is unstructured and contains two or three UIMs and a polyQ stretch . The UIMYOD1 is a conserved deubiquitinase belonging to the OTU family  and prefUSP7 or HAUSP belongs to the Ubiquitin Specific Protease (USP) family of deubiquitinating enzymes and is involved in various biological pathways, including apoptosis, transcription regulation, DNA replication, and neuronal development. It contains an N-terminal polyQ region, followed by a TRAF domain, a catalytic domain and a C-terminal UBL domain. USP7 has many different interactors, which can function as scaffolds, activity modulators, or substrates for deubiquitination. Well-known substrates for USP7-mediated deubiquitination include the tumor suppressor protein p53 and WASH, a protein that is essential for endosomal protein recycling. USP7 regulation and complex formation is reviewed elsewhere . USP7 inDrosophila. Surprisingly, overexpression of a catalytic-dead mutant resulted in the same protective effects. This indicates that the role of USP12 in HD pathology is independent of its deubiquitinase activity. In addition, no change in mHTT half-life was detected, which implies that USP12 is not involved in mHTT turnover. USP12 does lead to autophagy induction, which is needed for the neuroprotective effects of USP12 in HD. The authors suggest that autophagy could be involved by degrading a subpopulation of mHTT that is not measured in their half-life assay. It could also be that autophagy regulates HD pathology by degrading other substrates, and by improving general proteostasis, mHTT-induced toxicity is reduced  have been identified to influence HTT levels or the HD phenotype  and revivia the proteostasis machinery?In addition, different methods to analyze soluble and insoluble HTT protein levels are used in different studies. The mHTT protein can be present in different forms ranging from soluble monomers and High Molecular Weight (HMW) polymers to insoluble mHTT aggregates and IBs, and different methods can be used to study these different forms of mHTT. Many studies use fluorescently tagged HTT to quantify the number of IBs by microscopy, including determination of aggregate size, and this is different from biochemical assays using protein lysates. Here, lysate fractions can be divided into Triton-X100 insoluble aggregates, Triton-X100 soluble monomers, and HMW polymers, which can be detected with SDS-PAGE western blot, and it is important to quantify both HTT aggregates and soluble levels to get a complete picture of the effects on total protein levels. Is an intervention responsible for a shift between the soluble and insoluble fraction, or does it affect total protein levels in vitro experiments using isolated enzymes to in vivo experiments using small animal models. In cells, the experimental conditions are easily controllable, and it is relatively easy to manipulate enzyme levels. In vivo models, on the other hand, offer the potential to study the effects of enzyme modulation in an entire system and to study the effects on HD pathology. HD pathology only manifests later in life, indicating that aging processes are important in the etiology of the disease. These aging processes are not always present in immortalized cell lines, which could contribute to differences in experimental outcomes. This ‘young’ phenotype is especially present in iPSCs. The fact that most iPSC do not show aggregation already suggests a different proteostasis network, as suggested by via CHIP between astrocytes and neurons, as CHIP activity was inhibited in neurons but not in astrocytes . PROTACs consist of a target protein-binding site and a binding motif for E3s, connected by a linker. By binding to an E3 and a protein of interest, PROTACs improve the ubiquitination of their target, which results in improved degradation by the UPS . Several


Tumor-specific protein 70 (SP70) was identified as a new biomarker associated with the proliferation and invasion of cancer cells. This study aimed to investigate the expression of SP70 in hepatocellular carcinoma (HCC) and assess its clinical value in the diagnosis and prediction of early HCC recurrence.A total of 1049 subjects from the First Affiliated Hospital of Nanjing Medical University were recruited in this study. Serum SP70, alpha-fetoprotein (AFP) and prothrombin induced by vitamin K absence II (PIVKA-II) were measured. The diagnostic performance for HCC was obtained using the receiver operating characteristic (ROC) curve, and recurrence-free survival (RFS) was calculated using the Kaplan–Meier method. Univariate and multivariate analyses were performed to identify predictive factors of RFS.P<0.001). SP70 combined with AFP showed the best diagnostic performance . Kaplan–Meier analysis revealed that patients with high SP70 levels had shorter median RFS than those with low SP70 levels (P=0.003). In addition, high SP70 levels were significantly associated with shorter RFS (P=0.037) in the AFP-negative subgroup. Univariate and multivariate analyses confirmed that preoperative serum SP70 level, serum AFP, tumor diameter and microvascular invasion were independent prognostic factors of RFS.SP70 was highly expressed in HCC cells and HCC tissue. Serum SP70 levels in the HCC group were significantly higher than in the benign liver diseases group and healthy control group (SP70 is a promising biomarker in diagnosing HCC. High preoperative serum SP70 level is associated with an increased risk of early relapse and could be used as a valuable marker to predict early recurrence of HCC after resection. Liver cancer represents the sixth most common malignancy worldwide and the fourth leading cause of cancer-related death , 2. HepaAlpha-fetoprotein (AFP) has been the most utilized surveillance biomarker for HCC worldwide. However, its role has been controversial for decades due to the lack of sensitivity . In addiWe previously reported a new antigen with the relative molecular mass (Mr) of 70 kDa—tumor-specific protein 70 (TSP70 or SP70)—recognized by the monoclonal antibody (McAb) named NJ001. As a tumor derived antigen, SP70 is widely involved in the regulating the expression of numerous genes (GEO accession number: GSE59655), promoting cancer cell proliferation and metastasis. Serum SP70 levels were significantly correlated with tumor differentiation and lymph node metastasis and played an important role in cancer chemotherapy monitoring , 12. MorA total of 1049 subjects from the First Affiliated Hospital of Nanjing Medical University were enrolled in this study between October 2017 and March 2019, including 365 HCC patients, 310 patients with benign liver diseases (BLD) and 374 healthy volunteers. The BLD group included 101 patients with liver cirrhosis (LC), 100 with chronic hepatitis, 53 with hepatic hemangioma, 35 with hepatic cyst, 18 with hepatapostema and 3 with focal nodular hyperplasia. Among the HCC patients, 168 patients who underwent hepatectomy were followed up. Patients with HCCs were confirmed by pathology, and who had complete clinical data were included in the study. We excluded patients: 1) with other malignant tumors; 2) recurrent HCC; 3) who received preoperative anti-cancer treatment; and 4) who died during the perioperative period. All patients with benign liver diseases were followed up for 6 months after sampling to confirm non-HCC. The staging was assessed using the Barcelona Clinic Liver Cancer (BCLC) staging system.All blood samples were centrifuged at 3000 rpm for 10 minutes at room temperature for serum separation within 2 hours after collection. Serum SP70 was detected using an enzyme-linked immunosorbent assay (ELISA) kit . Serum AFP and PIVKA-II levels were measured on a Roche cobas e602 automated analyzer  and LUMIPULSE G1200 immunochemistry system , respectively.To determine the expression of SP70 in liver tissues, we collected HCC and corresponding adjacent tissues. Paraffin-embedded samples were sectioned at 4 mm thickness, and SP70 expression was detected with immunohistochemistry (IHC) kit (Code Biotech). All the specimens were independently examined by two investigators.2 at 37°C.Human HCC lines Hep3B, Huh7 and normal human hepatic cell L02 were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai. DMEM  supplemented with 10% FBS (Gibco) was used as the culture medium for all HCC cell lines. The RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco) was used for L02 cell culture. Cell lines were incubated in a humidified atmosphere containing 5% CO® 488A Antibody Labeling Kit  for fluorescence imaging analysis and the final concentration of NJ001-488A was 250 μg/mL. Briefly, cells were grown to 80% confluence on coverslips and then fixed in 95% ethanol at room temperature. After washing with PBS, the cell membranes were broken in 0.25% Triton X-100 for 15 min, blocked with 3% BSA for 2 hours at room temperature, and then incubated with diluent (1:50) NJ001-488A for 2 h at 37°C. Finally, the slides were washed with PBS three times and sealed with Antifade Mounting Medium with DAPI (Beyotime Biotechnology) and visualized using a Nikon Eclipse Ti fluorescence microscope .Direct immunofluorescence was performed as follows. A dose of 100 μg NJ001 was labeled with Mix-n-StainTM CFAll HCC patients who underwent curative resection were followed up. Liver ultrasonography and tumor marker assays were performed every 1–2 months after surgery. Suspected tumor recurrence in the liver was confirmed using enhanced computed tomography (CT) or magnetic resonance imaging (MRI) or biopsy. Recurrence-free survival (RFS) was defined as the time from curative surgery to recurrence.P-value of <0.05 was considered significant. Diagnosis-related indicators obtained from receiver operating characteristics (ROC) analysis, including sensitivity, specificity and the area under the curve (AUC), were used to assess the performance of tumor markers. The Cox proportional hazards regression model was performed to identify the independent predictive factors of RFS. A cut-off of P<0.05 was used to select variables entered into the multivariate model from the univariate analysis. Kaplan-Meier analysis with the log-rank test was utilized to compare RFS of different risk groups, and P<0.05 was considered statistically significant.All statistical analyses were performed with SPSS for Windows, version 22 . Data were expressed as median and interquartile range. Differences between categorical variables were assessed using the Pearson’s chi-squared test or Fisher exact test. Mann–Whitney and Kruskal–Wallis tests were used to assess differences between groups of nonparametrically distributed continuous variables. Bivariate correlations were evaluated using Spearman rank correlation coefficient. A P<0.05).The results of indirect immunofluorescence showed that SP70 was highly expressed in Hep3B cells, mainly expressed in the nucleus of Huh7 cells and lowly expressed in L02 cells (P<0.001). The median levels of SP70 were 11.4 ng/mL, 9.0 ng/mL and 6.0 ng/mL in the HCC, BLD and HC groups, respectively .To evaluate the potential application of SP70 in HCC, we divided the 1049 study participants into three groups. The HCC group had significantly higher SP70 levels compared with the BLD group and healthy controls (HC) , and there was no significant difference between SP70 and PIVKA-II (P>0.05). P>0.05). When distinguishing HCC from LC, the diagnostic performance of three markers decreased further. The AUC values were 0.627 (95% CI=0.569–0.685), 0.658 (95% CI=0.611–0.706) and 0.689 (95% CI=0.631–0.746) for SP70, AFP and PIVKA-II, respectively (P>0.05).To evaluate the diagnostic performance of SP70, AFP and PIVKA-II in HCC, we performed ROC analysis Figure 3P<0.001), combination of AFP and PIVKA-II  and PIVKA-II (P=0.002) alone. When distinguishing HCC from CH, an AUC value of 0.827 (95% CI=0.787–0.866) could be obtained by combining SP70 and AFP, which was significantly higher than a combination of AFP and PIVKA-II , combination of SP70 and PIVKA-II  and PIVKA-II (P=0.009) alone. Likewise, the combination of SP70 and AFP showed a better diagnostic value in differentiating HCC from LC  than the other two combinations (P<0.001).We combined different markers to enhance their diagnostic performance. Comparisons of different two-marker combinations are shown in P=0.003), P=0.037), A total of 168 cases were followed up after hepatectomy. None of the patients received postoperative adjuvant treatment. Twelve were lost to follow-up, giving a follow-up rate was 92.9%. During a median of 11 months of follow-up, 71 patients (45.5%) cases experienced HCC recurrence, including 64 (90.1%) with intrahepatic recurrence and 7 (9.2%) with extrahepatic metastasis. The 1- and 2-year recurrence-free survival rates were 62.3% and 33.2%, respectively. According to ROC analysis, the cut-off values of preoperative serum SP70, AFP and PIVKA-II for identifying early recurrence were 10.4 ng/mL, 20.94 ng/mL and 406 mAU/mL respectively. The clinical and biological features of HCC patients who underwent resection are available in P<0.001), tumor number , microvascular invasion , serum AFP  and serum SP70 . Multivariate analysis demonstrated that tumor diameter >5 cm , microvascular invasion , preoperative high-level SP70  and high-level AFP  were independent prognostic factors of RFS.The results of the univariate and multivariate analysis for RFS are shown in Despite being one of the most common malignancies with poor survival globally, HCC is limited in diagnosis and treatment due to its atypical early symptoms and high recurrence rate. Although guidelines from the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) recommend surveillance to be primarily imaging-based , 15, it In this study, we first reported the role of SP70 in the diagnosis and prognosis of HCC. As a new potential biomarker, SP70 has been proved to be valuable in lung cancer and gastric cancer detection in previous studies , 19, whiFor the diagnosis of HCC, AFP has high specificity but insufficient sensitivity. When using a cut-off level of 20 ng/mL, its sensitivities and specificities were 41%–65% and 80%–94%, respectively . AFP neeConsidering the limited accuracy of single indicator, it is often necessary to combine other markers to improve the diagnostic performance. In this study, combining SP70 with AFP or PIVKA-II significantly improved the performance compared with using SP70 alone. This may be attributed to the special expression mechanism of SP70 unrelated to the other two indicators. However, when used for detecting early HCC, the combinations did not display satisfactory results as expected. Additionally, the heterogeneity of assay methods in evaluating the performance of markers should not be ignored. Serum AFP was detected by electrochemiluminescence (ECL), and PIVKA-II was measured by chemiluminescence (CL). Due to their advantages of having a wide linear range, high sensitivity, rapid analysis with a low background and high automatization, ECL and CL have been broadly applied in biomolecule detection . In contde novo tumors arising in the cirrhotic liver (>2 years)  , 32. The2 years) . In this2 years) –37, whic2 years)  assessed2 years)  showed t2 years) . The exp2 years) –43. As aThis study is the first to demonstrate the diagnostic and prognostic role of SP70 in HCC, affirming its clinical predictive value and application prospect. Undeniably, our study has some limitations. This is a single-center study, that may lead to potential bias in specimen and data collection. Another limitation is that data on AFP-L3 detection is not provided in the comparison of markers. Finally, data on the characteristics of SP70 in patients with unresectable HCC and its assessment of overall survival were not provided. The follow-up period needs to be extended in further studies.In conclusion, SP70 is highly expressed on the membrane and in the cytoplasm of HCC cells. SP70 could be used as a valuable biomarker for HCC, and a combination of SP70 with AFP enhances diagnostic efficiency compared to individual biomarkers and a combination of AFP and PIVKA-II. Furthermore, SP70 is significantly and independently associated with RFS and could be a sensitive indicator in predicting early recurrence of HCC.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.Study design, guidance in writing of the manuscript, and review of the final manuscript: S-YP. performance of experiments: HS, Y-XJ. collection of the data: JW, W-XC, C-RG, YM and JX. analysis and interpretation of data: LW and HS. statistical analysis: LW and YM. writing of the manuscript: LW. All authors contributed to the article and approved the submitted version.


Van der Waals (vdW) ferroelectrics have attracted significant attention for their potential in next-generation nano-electronics. Two-dimensional (2D) group-IV monochalcogenides have emerged as a promising candidate due to their strong room temperature in-plane polarization down to a monolayer limit. However, their polarization is strongly coupled with the lattice strain and stacking orders, which impact their electronic properties. Here, we utilize four-dimensional scanning transmission electron microscopy (4D-STEM) to simultaneously probe the in-plane strain and out-of-plane stacking in vdW SnSe. Specifically, we observe large lattice strain up to 4% with a gradient across ~50 nm to compensate lattice mismatch at domain walls, mitigating defects initiation. Additionally, we discover the unusual ferroelectric-to-antiferroelectric domain walls stabilized by vdW force and may lead to anisotropic nonlinear optical responses. Our findings provide a comprehensive understanding of in-plane and out-of-plane structures affecting domain properties in vdW SnSe, laying the foundation for domain wall engineering in vdW ferroelectrics. Van der Waals ferroelectrics hold promising potential as electronic materials. Here, the authors have pioneered an electron microscopy technique to unveil both in-plane and out-of-plane structures critical to their switching behavior, providing valuable insights into potential device applications.  Compared with conventional FEs4, vdW FE materials possess weak interlayer interaction and demonstrate atomically thin ferroelectricity, moderate band gap, and dangling-bond-free interfaces, providing opportunities to design next-generation ultra-thin memory devices and sensors6. So far, various vdW FE materials have been experimentally demonstrated, including CuInP2S69, Peierls-distorted WTe212, In2Se314, bilayer h-BN15, and group-IV monochalcogenides 18. Among the reported FEs, 2D MX compounds show room-temperature in-plane polarization19 and giant nonlinear optical responses down to a monolayer limit21, which can be manipulated by tuning various parameters including electric field and strain24, making it a promising candidate for integrated device applications such as nonlinear optical and photocurrent switches25.Recent discoveries of two-dimensional (2D) van der Waals (vdW) ferroelectrics (FEs) are of particular interest due to the new phenomena and functionalities arising from the special atomic arrangements in combination with the ferroelectric order26, where the in-plane opposite displacements between the metal and chalcogenide atoms, such as Sn and Se  are commonly observed with antiferroelectric (AFE) stacking, i.e. in-plane polarization antiparallelly aligned across the neighboring vdW layers, leading to a zero net polarization. For simplicity, such a stacking is also referred to as “AB” stacking27. In comparison, the FE stacking (or “AC” stacking)27 in thin flakes contains parallel-aligned polarization that add up constructively. To date, both FE and AFE stacking orders have been reported in thin-flake samples of SnS29 and GeSe30 using cross-sectional TEM. Nevertheless, the domain dependent stacking order and their effects on domain wall properties are largely unexplored. The limited experimental results have motivated new approaches to revealing the strain and stacking across various domains in ultrathin MX compounds, particularly the heterogeneity manifested at distinct domain wall types.Despite these promising achievements, there is a lack of understanding on the lattice distortion and heterogeneities across the domain walls at the nanometer scale, which affects critical parameters related to the phase transition, domain distribution, and domain switching of FE materials, as well as the as-resulted performance of FE-material-based memories, sensors, and switches. MX compounds have a lattice structure analogous to phosphorene Se Fig. , are res31 (4D-STEM)  Fig.  to reveaEM) Fig.  and out-EM) Fig.  simultan 32 Fig. , our res 32 Fig.  and uniq 32 Fig. . This ph33 on mica substrate followed by a direct transfer to a TEM grid (details in Methods). We utilized 4D-STEM with the electron microscope pixel array detector (EMPAD)34 )34 Fig.  to colle33 Fig. S and at e33 Fig. S. Since w22  from its parent paraelectric phase , we observed an ~90° angle between parallel twin domains. Since the armchair direction in SnSe lattice is parallel to the electric polarization, the ~90° twin walls that we observed are consistent with previous works17. In addition, we observed a small lattice rotation (~5°) between red and blue (or yellow and purple) stripe domains  that intersects with the vertical stripes is wider, a relatively larger in-plane continuous lattice rotation is introduced to the junction area  as b and the width of the vertical domain (a1-) as a. We measured the deformed length, d, of each vertical stripe at the superdomain boundary by the image contrast in Fig. 1- deforms a1+ and generates “T” junctions  and the normalized deformed length, which is defined as the ratio between deformed width and the original width of each a1- domain (d/a) , we analyzed ~50 stripe intersections in SnSe and summarized the geometries statistically. For simplicity, we refer to the width of the horizontal stripe at the boundary  Fig. . We obse/a) Fig. . This ob/a) Fig. , which ib/a) between the horizontal and vertical domain stripes is continuously adjusted from 1:1 to 6:1. After a relaxation, the equilibrium domain structures show a transition from “T” junction structures in lower ratio cases to needle tips in higher ratios , the local elastic energy in the horizontal a1+ domains  needs to be compensated by the continuous rotation mechanism, which explains our observation of the lattice rotation spanning ~150 nm. Lastly, a large uniaxial strain is presented in a1- domains to form a lattice matched structure. And to reduce the strain energy, the domain width shrinks and forms the needle tip39.To further investigate how the lattice explicitly deforms, we further analyze individual localized regions from the 4D data at a “T” junction . The rotation maps show that the superdomain boundary contains three “T” junctions with similar geometry for each case  with width ratios (b/a) 1:1 or 2:1 are deformed periodically by the vertical stripes (a1-), as shown in Fig. 1+/a1- domain interface, and decay along the [−110] direction, inducing gradients in both built-in strain and lattice rotation , which agrees with the trend in experimental observations.Our phase field simulation results showed similar effects, where the horizontal domains , it may contain a mixture of AB and AC stacking. To quantitatively determine the ratio between AC and AB stacking, we simulated diffraction patterns of 10-unit-cell thick SnSe lattices that contain different ratios of AC and AB stacking orders using the kinetic diffraction theory model41 , indicating a ~90% AC stacking in FE stripe domains and a ~50% AB stacking in AFE domains, respectively  stacking but allowed in AB (or AFE) stacking Fig. . Since ol41 Fig.  and S17.l41 Fig. . The siml41 Fig. . We utill41 Fig.  indicatel41 Fig.  of the nl41 Fig.  shows twely Fig. . To furtely Fig. . The zooely Fig. , indicat21. We conducted SHG measurements on SnSe flakes and observed angle-dependent polarization across different regions, confirming the influence of stacking on nonlinear optical responses of these flakes , Thermo Scientific Chemicals) as the precursor. A quartz boat filled with SnSe powder was placed in the center of a single-zone tube furnace, and a piece of mica was placed downstream ( ~ 10 cm from the SnSe precursor) as the growth substrate. During the synthesis process, the PVD system was first pumped down to a base pressure of ~10 mTorr. Subsequently, the furnace was ramped up to 440 °C in 10 min and held for 45 min for SnSe growth. Afterwards, the furnace was opened for rapid cooling. A mixture of 65 sccm Ar and 5 sccm H2 was used as the carrier gas throughout the synthesis process. SnSe sample was then transferred onto TEM grid using PMMA as the supporting layer and sonication to release the sample from the original substrate. SHG imaging was conducted to select targeting flakes with ferroelectric domains.33. The 4D datasets of the SnSe thin flake were acquired at 300 kV. A 0.5-mrad convergence angle was used, leading to a ~ 2.44 nm probe size  probe diameter) and 4.88 µm depth of focus42 s42 Fig. . For a 3each diffraction pattern . To generate initial masks, we compute the mean diffraction patterns across the entire dataset Fig. S and subs23, we calculated it based on the equation |35, the four strain maps  are derived based on a selected basis 45 and projector augmented wave method for treating core electrons46. We used the Perdew-Burke-Ernzerhof (PBE) form of exchange-correlation functional within the generalized gradient approximation (GGA)47, plane-wave basis with a cutoff energy of 450 eV, and a Monkhorst-Pack48 k-point sampling grid of 8 × 8 × 1. Ground state structures of monolayer SnSe were obtained by fully relaxing both atomic positions and in-plane lattice parameters while keeping a vacuum region of 12 Å along the plane normal (i.e. the z axis) to avoid the periodic image interactions. The maximal residual atomic force for structural relaxation was set to be 0.02 eV Å−1, and the convergence criteria for electronic relaxation is set to be <10−6 eV. To understand ferroelectric and ferroelastic transitions, we carried out potential energy surface calculations by constraining the relative fractional x–y coordinates of Sn-Se pairs and the lattice parameter along z, and relaxing both cell parameters and shape in the x–y plane as well as cartesian coordinates of all atoms along z direction. For accurate potential energy surface calculation, we used a higher plane-wave cutoff energy of 520 eV, a denser k-point sampling grid of 12 × 12 × 1, a vacuum region of 15 Å, the maximal residual atomic force of 0.01 eV Å−1, and the convergence criteria for the electronic relaxation of <10−7 eV.Atomistic and electronic structures of monolayer SnSe were calculated using first-principles density functional theory (DFT)Pi as the order parameters to describe the ferroelectric state. The equilibrium domain structure is determined by minimizing the total free energy (F) with respect to Pi via solving the time-dependent Landau–Ginzburg–Devonshire (LGD) equations,L represents the kinetic coefficient related to the domain wall mobility, t is time, r is the spatial position. The total free energy F includes the Landau, gradient, elastic, and electrostatic energies,εij and Ei represent the components of strain and electric fields. Since the magnitude and in-plane orientation of spontaneous polarizations in 2D SnSe ferroelectric materials and PbTiO3 are similar49, in this simulation, we use a six-order Landau polynomial function for SnSe 2D ferroelectrics, and select the corresponding Landau energy coefficients to be same as in PbTiO3. Detailed expressions of each free energy density in Eq.  is chosen to be 50. To mimic the 2D in-plane ferroelectric domain structure, the substrate strain is set to be 5% to keep the global a1/a2 structure stable. The Landau coefficients, electrostrictive coefficients, and elastic-compliance constants are collected from Ref. 49.In the phase-field simulations, we use polarization vector entclass1pt{minima41 package with the same conditions.ADF-STEM image is taken in 300 kV, and the dwell time is 10 µs at each scan position. The convergence angle of the electron probe is 25 mrad, with a 115 mm camera length. The simulated ADF-STEM images are generated by the multi-slice method in abTEMSupplementary InformationPeer Review File