Monoclonal antibodies (mAbs) are the fastest-growing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases. Aggregation of mAbs continues to be a major problem in their developability. Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomer-monomer association followed by nucleation and growth. Although the aggregation propensities of antibodies and antibody-based proteins can be affected by the external experimental conditions, they are strongly dependent on the intrinsic antibody properties as determined by their sequences and structures. In this review, we describe how the unfolding and aggregation susceptibilities of IgG could be related to their cognate sequences and structures. The impact of antibody domain structures on thermostability and aggregation propensities, and effective strategies to reduce aggregation are discussed. Finally, the aggregation of antibody-drug conjugates (ADCs) as related to their sequence/structure, linker payload, conjugation chemistry and drug-antibody ratio (DAR) is reviewed. F increasing and ΔGU-F decreasing) or the  unfolded structure becomes stabilized (GU decreasing) . Th. Th56].  and Gln . VH and able Fab . Fab con glycans . In cont9-Lys409 . CH2 assL is packed against the four-stranded β-sheet of the CH1 domain, thus mediating aggregation [IgG contains extensive intra-domain and inter-domain hydrophobic interactions. When subjected to structural fluctuations, those hydrophobic interactions are readily exposed to constitute aggregation nuclei . The subregation . The intregation . Besidesregation . In thisregation . On the regation . Alternaregation . Anotherregation .H2 and Ile253 in the CH2–CH3 junction [Another factor complicating IgG aggregation is the glycosylation. Usually, the glycans attached at N297 are believed to benefit the aggregation resistance for IgG by shielding hydrophobic residues from being exposing to the solvent. Trout et al. have used SAP calculations to demonstrate that glycan attached at IgG1 Asn297 shields the couples of hydrophobic residues, such as F241 and F243, from exposure to the solvent. Consequently, the aglycosylated mAbs are less stable and therefore aggregate more easily than the glycosylated mAbs . In addijunction . Althougjunction ,70. In mjunction . Thus, mjunction ,72.H/VL, CH1/CL, CH2/CH3 and CH3/CH3 interfaces. The domain-domain interactions confer IgG interface free energy for native folding, which would disappear when one of the interacting domain unfolds [H2 unfolds first and triggers the aggregation process, while CH3 is the most stable domain [On the other hand, the domain-domain associations need to be taken into account when studying the aggregation of IgG, which contains substantial molecular-scale interactions in the V unfolds . Thus, te domain . Fab usue domain . IgG agge domain . The lowe domain . Conseque domain . The lowe domain . The DSCe domain ,75, whicH and VL as single domains are the smallest units for antigen binding. These single domains have attractive advantages as diagnostic and therapeutic reagents due to their small size, which confer them capacity to access cryptic epitopes and enhance penetration into solid tissues [H and VL are intrinsically prone to aggregation due to the exposure of the hydrophobic VH–VL interface. VH has been shown to be more prone to aggregation than VL, and its aggregation is more dependent on its CDRs than VL [H and VL results from the hydrophobic core inside the immunoglobulin β barrel and the disulfide bond connecting the two β sheets layers. While an intra-domain disulfide in the antibody domain contributes a range of 4–6 kcal/mol of free energy to the folding [2 of the hydrophobic contact corresponds to 25 cal/mol [H and VL. Generally, the aggregation resistance strategies could be divided into rational and evolutionary approaches. While rational approaches exploit structure-based knowledge or sequence information to guide the aggregation-resistant mutations, the evolutionary methods involve the selection of a single domain antibody library by stability-improving pressures, such as temperature and pH. These two approaches could be combined to design aggregation-resistant single domain antibodies (sdAb)  .H–VL interface and the residues in framework (FR) regions influencing aggregation. Antibodies with net charges are less prone to aggregation due to the intermolecular repulsions. Tanha et al. have reported that the aggregation-resistant human VHs obtained by panning against target enzymes using a transient heat denaturation approach bear acidic pIs, similar to the naturally-occurring camelid VHH and shark VNAR, which is reminiscent of protein acidification constituting a universal mechanism to confer functional sdAbs [H and VL. Perchiacca et al. have improved the solubility of VH containing aggregation-prone Aβ peptides by inserting the “DED” triad into the edge of CDR3 [H  and CDR2 of VL , are universal strategies for conferring aggregation resistance to sdAbs [H and VL. Kim et al. have reported that the introduction of the non-canonical disulfide bond between Cys54 and Cys78 residues increased the thermostability of VH by 14–18 °C [L by 5.5–17.5 °C through the creation of disulfide bonds linking residues 48 and 64 [H–VL interface that would otherwise be buried in intact antibodies or other bigger fragments. Indeed, hydrophilic mutations near the former VH/VL interface have been demonstrated to improve the solubility of dAbs [HH from camels has inspired the screening of aggregation-resistant mutants of VH, although the camelized mutations are often not applied to therapeutic mAbs considering the potential immunogenicity [H and residues of R24, Y49 [L are the key players in mediating the aggregation of sdAbs.The elucidation of structural and sequence determinants underlying aggregation propensity diversifies the rational approaches of combatting the aggregation of sdAb, which include the engineering of the overall charge, CDRs, disulfide bonds, Val sdAbs . The samal sdAbs . Consist of CDR3 . Dudgeonto sdAbs . In addito sdAbs ,82 and tto sdAbs ,83,84. O14–18 °C . In addi8 and 64 . Another of dAbs . The natgenicity . Furthergenicity , Arg66 agenicity  in VH anR24, Y49  and Pro8R24, Y49  in VL arH1, 3 and 5 gene families generally displayed better physical characteristics than the 2, 4 and 6 gene families [L, Vκ is more desirable than the Vλ chain, with Vκ3 being the most thermodynamically-stable followed by the Vκ1 and Vκ4 [H, which usually bear high frequencies in the repertoire [H antibody, m36, for targeting HIV-1 CoRbs by panning a VH phage library, which is composed of a stable scaffold based on the VH3 with the incorporation of partially-randomized CDR1 (Positions 30 and 31) and naturally-occurring CDR2 and CDR3 [Alternatively, the library-based evolutionary approaches could be used for alleviating the aggregation of sdAbs. Antibody domains of different sub-families bear distinct thermostability and aggregation propensity. The Vfamilies . For VL, and Vκ4 . Besidespertoire . By combpertoire . Our groand CDR3 . On the and CDR3 .H and VL connected by a flexible linker, which combines with the strong inter-domain associations to ensure that scFv retains the antigen binding of the full-length IgG if no reorientation of the VH and VL occurs. Due to its small size (~30 kD), scFv bears many therapeutic merits as a diagnostic and therapeutic agent, such as easy expression, relative stability compared to VH and deceased toxicity when used in radioisotopes and bio-imaging [H and VL, the thermostability and aggregation liability of scFv relates tightly to the CDRs’ sequences and the intrinsic stabilities of the sub-domains [H and VL are also applied to the scFv. General strategies include introducing the stabilization mutations, such as residues 6 and 66 in VH, Pro8 in Vκ [H and VL, such as the combinations of H3κ3, H1bκ3, H5κ3 and H3κ1 for constructing scFv [H and VLV Section). However, as a fragment with strong VH/VL associations, scFv has its unique features. The VH/VL interface of scFv is usually conformationally dynamic and experiences “transient opening”, which could expose the hydrophobic patches to promote aggregation [H–VL interface has enjoyed many successes for improving the solubility of scFv. Corresponding strategies include introducing a disulfide bond into the contact interface [H–VL [H domain of one scFv pairs with the VL domain of another scFv and vice versa [H and VL by changing both the linker sequences and length could alleviate the aggregation of scFv [scFv consists of the variable domains of V-imaging . Like th-domains . Thus, to8 in Vκ ,91, chooing scFv  and intrregation . Therefonterface , formingce [H–VL  and addice [H–VL . Anotherce versa . This doce versa . Thus, o of scFv . Includi of scFv . Besides of scFv .H, CH1, VL and CL, in which VH/VL and CH1/CL strongly associate via hydrophobic contact and hydrogen bonding, while few interactions exist at the VH/CH1 and VL/CL interface. Fab has unique advantages as the diagnostic tool and pharmaceutic reagent due to its small size, easy production and relatively longer in vivo half-life than the single domain antibody [H and scFv since the hydrophobic interfaces exposed in VH and scFv are buried by the constant domain in Fab. Like sdAbs and scFv, the unfolding and aggregation propensity of Fab are also impacted by CDRs. It is reported that IgG pools from human blood exist as dimers due to the association of the distal ends of their Fab arms, similar to the idiotype-anti-idiotype complexes [H1 and CL of IgG1 can lock domain conformations and strengthen the inter-domain interactions. By mimicking this disulfide bond, Peters et al. have improved the thermostability of Fab and the disulfide bond heterogeneity of full-length IgG4. This disulfide bond was formed through mutating C127 in the N terminus of CH1 to a serine and simultaneously introducing a cysteine at the C terminus of CH1 (Positions 227–230), which facilitates disulfide bonding to the CL [Fab contains four domains, namely, Vantibody . Fab-basantibody . Generalomplexes . The proomplexes . Besidesomplexes . Meanwhiomplexes . Anothero the CL .H/VL and CH1/CL in Fab is highly predisposed in the unfolding process, as shown by the single and sharp endotherm peaks in the DSC thermograms [H1/CL interface is believed to be more stable than the VH/VL interface, but also depends on the characteristics of CDRs. While the VH/VL interface is pre-defined for a given Fab, which has limited spaces for engineering considering the requirement of attaining the antigen binding, the CH1/CL interface constitutes a convenient platform for improving inter-domain interactions. Teerinen et al. have reported that the solvated hydrophobicity of CH1/CL could be increased by mutating Thr178 to Val or Leu, which leads to increased unfolding free energy of Fab [H1/CL interface including S64E, S66V in CH1 and S69L, T71S in Cκ. These mutations conferred CH1/CL with improved inter-domain associations due to enhanced hydrophobic contact and hydrogen bonds [On the other hand, unlike sdAbs, Fab structurally features two inter-domain interactions, which are targets for aggregation-resistant strategies. The domain-domain cooperativity of Vrmograms . The CH1y of Fab . Recentlen bonds . The impH2–CH3 interface of Fc. Indeed, the hydrophobic lower hinge (sequence: 231-APELLGGPSVFLFPP-245) is not only the “hot” site for engineering IgG effector function, such as antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) [H2 domain as revealed by the DSC analysis [H2 domain and decrease aggregation propensity by removing the unstructured loop composed of sequence of N-APELLGG-C [H2 could be significantly decreased by mutating APRs residues, such as F241, F243 identified by the TANGO program into hydrophilic residues . Another hot spot for combating aggregation lies at the CH2–CH3 interface, which bears high adaptability to binding different ligands, such as protein A/G, FcRn and rheumatoid factor [H2–CH3 junction contained a hydrophobic consensus motif composed of Asn434, Ile253, Met423, Tyr436, Met252 and Ser254 for accommodating different natural scaffolds, such as two α-helices of the B-domain of protein A and two β-strands (engineered peptide with sequence of DCAWHLGELVWCT) [H2–CH3 interface interact with the C’E loop (the residues Arg293–Thr299) containing glycosylated Asn297 and another nearby loop of the same neighboring molecule [H2 due to the interactions between F295 and GlcNAc1 and core fucose, it may be interesting to explore how the altered C’E loop impacts the Fc–Fc interactions and subsequent colloidal aggregation of IgG1.The hydrophobic regions contain APRs promoting antibody aggregation. For IgG, more APRs are found in Fc than in Fab, as evidenced by the molecular simulations performed by Bernhardt et al. . By usinty (CDC) , but alsanalysis . Gong etPELLGG-C . Furtherd factor . DeLano GELVWCT) . By mutamolecule . This comolecule . AlthougH2 and CH3. Actually, CH2 is considered to be the least stable domain and usually triggers the unfolding of full-length IgG [H2 and CH3 could benefit the stability and solubility of the full-sized IgG. One effective strategy for improving the stability of CH2 and CH3 is modulating intra-domain disulfide bonds. Gong et al. have increased the transition melting temperature (Tm) of CH2 from 54.1 °C to 73.8 °C by introducing a disulfide bond in Positions 242 and 334 (m01) [m of a monomeric CH3 from 41 °C to 76 °C by introducing a disulfide bond connecting Positions 343 and 431 [H3 domain. In their research, two engineered disulfides with one linking the N-terminus of the CH3 domain with the F-strand and the other connecting the BC loop and the D-strand collectively led to an increase of the Tm of ~15 °C for IgG Fc [Fc aggregation depends on the thermostability of Cngth IgG . Therefo34 (m01) . Ying et and 431 . Wozniakr IgG Fc . CollectAntibody drug conjugates (ADCs) combine the specificity of monoclonal antibodies (mAbs) with the potent cytotoxic activity of small molecule drugs for the treatment of cancer and other diseases . The devADCs have three major components, antibody, linker and the cytotoxic drug as the payload. Clearly, the aggregation propensity of ADC is directly related to the parent mAbs, as exemplified by Beckley et al. showing that eight kinds of ADCs with the same linker-payloads of vc-MMAE (monomethyl auristatin E), but with distinct variable domains had different propensities of forming high molecular weight species (HMWS) . MeanwhiFor the impact of the linker-payload, their hydrophobically-aliphatic moieties expand the APRs on ADC, facilitating the aggregation by forming hydrophobic patches. Guo et al. have pointed out the contributions of linker payload to the overall hydrophobicity of the ADC by molecular modeling . The incH2–CH3 interface compared to the parent antibody [H2 domain became more solvent exposed in ADC than in the naked mAb by using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The conformation alteration of CH2 domain was also confirmed by Beckley et al., showing that the CH2 domains in ADC with high DAR bear low stability and rapidly form aggregates at 40 °C [The one-pot chemical conjugation of mAb and drug reactants usually produces heterogeneous ADC products. In the case of thiol-maleimide chemistry, the extent to which the inter-chain disulfide bonds were reduced determined the quantity of drugs attaching to mAb, which led to the ADC species with different DARs ranging from 0 to 8. DAR constitutes a major concern for designing ADC therapeutics, since DAR tightly impacts the aggregation propensity, in vivo potency and serum stability of ADC. ADC with high DAR usually has higher aggregation propensity due to the increased hydrophobicity conferred by the hydrophobic drug molecules. Guo et al. have found that their ADC with DAR6 species may exist in a multimeric state, while DAR2 and DAR4 species likely exist in monomeric forms under ambient conditions . Beckleyantibody , which mat 40 °C . The facat 40 °C ) studiesCollectively, the aggregation of ADC is a function of the naked antibody, linker payload, conjugation chemistry and DAR, as well as the formulation solution, all of which need to be considered when trying to increase the aggregation resistance of ADCs.H1/Cκ heterodimer interface (S64E/S66V of CH1 and S69L/T71S of Cκ) identified from the Fab-like format could be applied onto the full-length IgG-like format (4Dm2m) [H2 domain through interaction with glycan could also confer full-sized IgG decreased low pH-induced aggregation [H and CDR2 of VL into negatively-charged residues, such as Asp and Glu, could successfully be transferred into scFv format [The aggregation of antibody domains has been extensively studied in order to expedite the screening of aggregation-resistant IgG in the early development process. However, caution is needed since aggregation resistance strategies for antibody domains are not always successfully transferred onto full-sized IgG. Usually, the strategies for improving the stability of the constant domain could be applied to full-length IgG. For example, our group has found that the stabilization mutations at the C (4Dm2m) . Kelly eregation . By contregation . Anotherv format . The scFms was significantly compromised as measured by the temperature ramping CD spectra. Meanwhile, mitigating aggregation through engineering the IgG sequence and structure by mutagenesis bears risks to generate new B- and T-cell epitopes and to cause immunogenicity. While the mutations occurring at CDRs could induce an anti-idiotype response and lead to the neutralization of the therapeutics, mutations at the constant domain could evoke the “binding antibodies” response in the host and compromise the efficacy of therapeutics due to the changed pharmacokinetics [One should also keep in mind that the same forces promoting protein aggregation also operate in protein folding and interactions. As such, one needs to balance between decreasing aggregation and maintaining the correct folding and specific binding when rationally-designing aggregation-resistant antibodies. In our development of aggregation-resistant m36.4 variants, we have found that although some mutants indeed gained decreased aggregation according to the dynamic light scattering (DLS) profiles, their unfolding Tkinetics . Thus, tkinetics . An effekinetics . Besideskinetics ,141.The antibody multi-domain nature defines its aggregation process as pathway dependent, which is a function of a combination of IgG sequences and structures, as well as the solution conditions. While most designs focus on improving thermostability and alleviating the aggregation of antibody fragments in a lumped assay, such as turbidity and dye binding, few studies are dedicated to clarifying to what extent the engineering strategies impact the formation of the aggregate intermediates or the soluble irreversible aggregate species. Similarly, few existing computational programs account for the aggregation mechanism of the antibody although they could predict APRs in antibody sequences. Furthermore, calculation tools have failed to incorporate the external solution condition into the aggregation prediction. Although several programs have indeed considered the physiological conditions in the prediction, therapeutic antibodies are not usually produced, stored and administered under physiological conditions. Thus, these calculation methods poorly learn the antibody aggregation-resistant engineering in some cases. The studies of soluble aggregate intermediates, aggregation kinetics and mechanisms, as well as their dependence on the solution conditions would guide the antibody formulation optimization, which should be extensively pursued in future research work.In summary, the aggregation propensities and aggregation-resistance strategies for antibody domains have been extensively investigated. Future studies are worth understanding the aggregation mechanisms for full-sized antibodies and their dependence on the environment, which could expedite antibody therapeutics development.


Phyllanthus niruri (50% ME of P. niruri) has alleviated NAFLD in Sprague–Dawley rats. This study aimed to assess the molecular mechanisms of action, and to further evaluate the antiangiogenic effect of this extract. NAFLD was induced by eight weeks of high-fat diet, and treatment was applied for four weeks. Antiangiogenic activity was assessed by aortic ring assay and by in vitro tests. Our findings demonstrated that the therapeutic effects of 50% ME among NAFLD rats, were associated with a significant increase in serum adiponectin, reduction in the serum levels of RBP4, vaspin, progranulin, TNF-α, IL-6, and significant downregulation of the hepatic gene expression of PPARγ, SLC10A2, and Collα1. Concomitantly, 50% ME of P. niruri has exhibited a potent antiangiogenic activity on ring assay, cell migration, vascular endothelial growth factor (VEGF), and tube formation, without any cytotoxic effect. Together, our findings revealed that the protective effects of P. niruri against NAFLD might be attributed to its antiangiogenic effect, as well as to the regulation of adipocytokines and reducing the expression of adipogenic genes.The growth of adipose tissues is considered angiogenesis-dependent during non-alcoholic fatty liver disease (NAFLD). We have recently reported that our standardized 50% methanolic extract (ME) of  Non-alcoholic fatty liver disease (NAFLD) is an emerging metabolic disorder that encompasses a wide range of liver injuries, extending from simple steatosis to non-alcoholic steatohepatitis (NASH) which can develop to fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC) .Liver is an essential organ for normal lipid homeostasis . Free faNAFLD is frequently correlated with obesity and insulin resistance, at the level of adipose tissue and liver. The adipose tissue in this condition becomes insensitive to the antilipolytic influence of insulin, which leads to an increase in the amount of released fatty acids. The β-oxidation of FFAs in the liver is declined . MoreoveThe de novo lipogenesis and uptake of FFA are mainly controlled by SREBP-1c gene (sterol regulatory element binding protein-1c) and carbohydrate responsive element-binding protein (ChREBP). Both SREBP-1c and ChREBP activate the expression of key biosynthetic enzymes specific for fatty acid synthesis, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) . In addiMany recent studies proposed that dysfunction in adipose tissue has been linked to the pathogenesis and progress of NAFLD. The lipotoxicity of accumulated fats in the liver triggers secretion of pro-inflammatory adipocytokines . MoreoveTaking into consideration the vital role of specific genes and adipocytokines on hepatic steatosis, fibrosis, and inflammation, researchers worldwide have strengthened their efforts to study the mechanistic pathways underlying the effects of therapeutic agents on adipocytokines and genes which are thought to contribute in NAFLD progression.Phyllanthus niruri (P. niruri) belongs to the Euphorbiaceae family, and grows in tropical and subtropical regions of the world. Due to its wealthy medicinal properties, it has been used extensively for more than 2000 years by different nations, such as Malaysia, and in Unani medicine and Indian Ayurvedic therapeutics for stimulating liver in treatment of jaundice, asthma, and bronchitis [P. niruri extract has shown potent hepatoprotective and antioxidant effects, and antihyperlipidemic activities [onchitis . P. nirutivities ,13.P. niruri inhibited the progression of NAFLD in Sprague–Dawley (SD) rats, reduced NAFLD activity score (NAS), prevented fibrosis, and improved serum levels of liver enzymes, glucose, insulin, insulin resistance, lipid profile, serum free fatty acids (FFAs), and hepatic contents of total cholesterol and triglyceride [P. niruri against NAFLD, as well as to study the expression of some genes by which P. niruri extract have been expected to exert its activities. We also aimed to further investigate the antiangiogenic activity of 50% ME of P. niruri in vitro.Despite our earlier encouraging data, which confirmed that a standardized 50% methanolic extract (50% ME) of lyceride , to dateP. niruri were collected and a voucher specimen (No. 11474) was kept in the Herbarium Unit at School of Biological Sciences, Universiti Sains Malaysia. The plant was rinsed with clean water, and dried, then was ground into powder. The dry powder was macerated by continuous stirring with 50% methanol/water at 40 °C for 48 h, and then evaporated under reduced pressure and completely dried. The dried extract was stored at 4 °C until further use.The whole plant parts of n = 6). Group (1), the normal control group, was fed a normal diet for 8 weeks, while the other three groups  were fed with HFD for 8 weeks. Treatment was applied from week 5. Group (1) was treated with distilled water (10 mL/kg body weight). Group 2, the negative control group, was treated with distilled water (10 mL/kg body weight); group 3, the positive control group, was treated with metformin (500 mg/kg body weight), and group 4 received the treatment of 50% ME of P. niruri (1000 mg/kg body weight).In total, 24 male Sprague–Dawley (SD) rats aged (10 weeks old) were provided by the Animal Research and Service Centre, Universiti Sains Malaysia. All experimental protocols and methods were performed in accordance with the relevant guidelines and regulations of the Experimental and Animal Ethics Committee of the School of Pharmaceutical Sciences, Universiti Sains Malaysia (protocol No. 2013/(90) (546)). Rats were fed with a high-fat diet (HFD) for 8 weeks to induce NAFLD, as previously described . Animalsv/v) for histological analysis, while the other part was immediately kept in RNA later solution , and stored at 4 °C overnight to prevent RNA enzymatic degradation. Then, all samples were placed in the freezer at −80 °C until further use for gene expression analysis. Serum samples were used to determine hepatic liver enzymes.At the end of week 8, rats were fasted overnight and anesthetized. Blood was collected via cardiac puncture, and centrifuged to get the serum, which was stored at −80 °C until further use. Liver samples were removed from the largest hepatic lobe, washed with a chilled 0.9% NaCl solution, dried, and weighted. Then, liver samples were divided into two parts: one part was fixed in formaldehyde 10% (Liver sections were embedded in paraffin and stained with hematoxylin and eosin (H&E) stain to evaluate steatosis and inflammation, and also stained with Masson’s trichrome for confirmation of fibrosis.P. niruri and metformin treatment on serum adipocytokines in NAFLD-induced rats, serum adiponectin, RBP4, and progranulin were measured by rat ELISA kits  according to the manufacturer’s protocols. Serum TNFα, IL-6, and vaspin concentrations were determined by sandwich enzyme immunoassay technique (ELISA) .To evaluate the effect of 50% ME of Liver specimens collected from sacrificed rats were kept immediately in the preservative RNA later solution.  at 4 °C overnight, and then were stored in −80 °C until used. Approximately 30 mg of the frozen liver tissues were mixed with lysis buffer and homogenized. Total RNA was extracted using RNeasy plus Mini Kit  according to the manufacturer’s protocol.RNA purity was measured spectrophotometrically via a NanoDrop ND-2000c Spectrophotometer , and the ratio of the readings at 260 nm and 280 nm (A260/A280) was estimated. RNA integrity was quantified by using Agilent 2100 bioanalyzer .RNA was reverse-transcribed to cDNA using Transcriptor First Strand cDNA Synthesis Kit , according to the manufacturer’s protocol. Concentration of the resulting cDNA was measured spectrophotometrically, and was amplified using TaqMan rat assay genes from Applied Biosystems . The amplification reaction was performed using StepOnePlus Real-Time PCR System .Quantitative Real-Time PCR (qRT-PCR) was carried out for the following genes: peroxisomal proliferator-activated receptor-γ (P PARγ) (NM_001145366.1), solute carrier family 10 (sodium/bile acid cotransporter) member 2) (SLC10A2) (NM_001270862.1), complement factor D (adipsin) (CFD) (NM_001077642.1), patatin-Like phospholipase domain containing 2 (PNPLA2) (NM_001108509.2), collagen alpha 1 (Coll α1) (NM_053304.1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (NM_017008.4) and hypoxanthine phosphoribosyl transferase (Hprt1) (NM_012583.2) were used as housekeeping genes . All mea−ΔΔCt method [The amount of target transcript in each sample was normalized to GAPDH and HPRT-1, and the averages of the obtained threshold cycle (Ct) values were processed for further calculations according to the comparative Ct method. Fold change was determined according to the arithmetic formula 2t method  and ΔΔCTΔCT represents the difference in Ct value between the targeted gene and the housekeeping genes.P. niruri whole plant extract [P. niruri treatment groups was calculated by comparing length of blood vessels in control (0.5% dimethyl sulfoxide (DMSO)) and treated rings (P. niruri).A standard method was used to screen the antiangiogenic potency of 50% ME of  extract . Photos P. niruri against somatic endothelial cells (EA.hy926) were studied using MTT cell viability assay following a standard method [The antiproliferative effects of 50% ME of d method ,18. Abso50 value of P. niruri was determined from the logarithmic regression equation by plotting a graph between different concentrations and mean percent inhibition of cell proliferation. After determination of IC50, all the other experiments were carried out using three different concentrations i.e., IC25, IC50, and IC90 of P. niruri. Values shown are mean ± SD of three independent experiments.The ICP. niruri against EA.hy926 cells [n = 3).The scratch assay was used to study the anti-migratory effects of 50% ME of 26 cells ,20. MediP. niruri extract to arrest the differentiation of EA.hy926 cells into tube-like structures on Matrigel matrix was evaluated using a reported method [P. niruri treated cells [n = 3).The ability of 50% ME of d method . DMSO aned cells . The resP. niruri on the levels of VEGF secreted from human colon cancer HCT 116 cells. Media with 0.5% DMSO was used as negative control. The R2 value obtained from linear regression equation of VEGF standard calibration curve was used to calculate the concentration of VEGF in the 0.5% DMSO-treated and P. niruri-treated cells, and the percent inhibition of VEGF secretion in P. niruri-treated cells was calculated using a reported equation. The values presented are in mean ± SD (n = 3).Human VEGF Duoset ELISA kit  was used to determine the effect of 50% ME of http://www.multid.se) [p < 0.05.The obtained data were expressed as means ± SEM. Statistical significance was analyzed using version 20 of the Statistical Package of Social Sciences (SPSS) statistical program . Real-time PCR results were analyzed using GenEX5 program (ltid.se) , and folP. niruri extract and metformin clearly reduced steatosis and prevented fibrosis.Histological changes in liver sections from each group are presented in p < 0.01) reduction in serum adiponectin levels in HFD group (7.80 ± 0.63 µg/mL) compared with normal group (15.05 ± 1.49 µg/mL). However, the concentration of serum adiponectin was significantly increased (p < 0.01) after the treatment with P. niruri (13.93 ± 1.28 µg/mL) compared with HFD group, while serum adiponectin level was not improved in rats treated with metformin. On the other hand, serum levels of both TNFα and IL-6 were significantly (p < 0.05) increased in HFD group compared with normal diet group, while the treatment with P. niruri and metformin significantly inhibited this elevation in circulating levels of serum TNFα (p < 0.05) and IL-6 (p < 0.01) compared with HFD group. Moreover, there was a considerable increase in the serum levels of RBP4 (p < 0.01) in the high-fat diet group (13.28 ± 0.86 µg/mL) compared with normal diet group (6.61 ± 0.49 µg/mL). However, this trend was significantly (p < 0.01) reversed in rats treated with P. niruri (7.50 ± 0.77 µg/mL) and metformin (7.80 ± 0.49 µg/mL) groups.As shown in , the conp < 0.01) higher in the HFD group (429.43 ± 30.38 ng/mL) and it was non-significantly (p = 0.065) lowered (358.15 ± 15.21 ng/mL) after the treatment with P. niruri, and significantly (p < 0.05) reduced in rats treated with metformin (340.37 ± 22.68 ng/mL).Serum concentration of progranulin was significantly (p < 0.01) elevated in the HFD group (739.11 ± 22.23 ng/mL) compared with the normal control group (407.65 ± 17.49 ng/mL). However, this value was significantly (p < 0.01) decreased in the group treated with P. niruri (595.72 ± 12.19 ng/mL) compared with the HFD group, however, surprisingly, this value was even significantly higher (p < 0.01) in metformin-treated group (884.02 ± 12.35 ng/mL).The concentration of serum vaspin was significantly  was checked via Agilent 2100 Bioanalyzer, and demonstrated that RIN ranged between 7 and 10 for all samples, and the apparent ratio of 28S to 18S ribosomal RNA was approximately 2:1 (data not shown), suggesting that our extracted RNA samples were intact and could be used for real-time PCR.p < 0.01) upregulation of three target genes , compared to the calibrator , while causing insignificant downregulation in PNPLA2 gene (0.45 ± 0.07) and upregulation in CFD gene (2.01 ± 0.14).The expression levels of PPARγ, SLC10A2, CFD, PNPLA2, COL1α1, and the two endogenous reference genes were subsequently validated by qRT-PCR measurements. All genes showed that the efficiency was between 90–110%, and the slope between (−3.1) and (−3.5), which are within the reference criteria to run the qRT-PCR. After data analysis of the Ct values by GenEX5 program and normalizing to the reference genes HPRT-1 and GAPDH, all measured mRNAs showed a different expression level between calibrator group  and negative group (HFD group). Findings showed that high-fat diet caused highly significant (P. niruri-treated group were significantly downregulated by 20.5 ± 8.6 (p < 0.05)-, 7.3 ± 2.1 (p < 0.01)-, and 5.9 ± 1.8 (p < 0.01)-fold, respectively, compared with the expression in HFD group. Additionally, the expression of these genes in metformin-treated group were also significantly downregulated by 18.5 ± 8.7 (p < 0.05)-, 9.5 ± 2.2 (p < 0.01)-, and 6.7 ± 1.8 (p < 0.01)-fold, respectively, in comparison to HFD group , whereas the fold change values in FD group .P. niruri extract  of dose-response curve, was 28.38 ± 0.82 µg/mL. Suramin, at the concentration of 100 µg/mL, caused 90% inhibition of blood vessel outgrowth.Significant inhibition of blood vessel outgrowth was observed in thoracic rat aortic rings treated with different concentrations of  extract . PercentP. niruri whole plant extract did not induce cytotoxicity towards EA.hy926 cells at the tested concentrations (100–3.125 µg/mL). The IC50 of 50% ME of P. niruri extract was calculated to be 136.26 ± 7.76 µg/mL. Based on the IC50 value of 50% ME of P. niruri towards EA.hy926 cells, all further in vitro assays were conducted using three different concentrations . P. niruri against EA.hy926 cells. No apparent changes in morphology of EA.hy926 cells were observed in P. niruri extract treatment groups.The MTT cell viability assay revealed that 50% ME of P. niruri treatment groups. Percent inhibition of cell migration after 12 h of treatment with IC25, IC50, and IC90 concentrations of P. niruri extract was 73.50 ± 4.63%, 85.26 ± 12.21%, and 93.50 ± 6.04%, respectively. P. niruri extract towards EA.hy926 cells.Dose-dependent reduction in migration potency of EA.hy926 cells across the artificially created wound was observed in 50% ME of P. niruri treatment groups. P. niruri extract caused significant reduction in the percentage of area occupied by the formed tubes, total length of tubes, number of loops formed, and number of branches, separately. Pecentage inhibition of tube formation at IC25, IC50, and IC90 of P. niruri extract was 35.58 ± 16.30%, 71.07 ± 4.78% and 87.58 ± 1.47% respectively, while percent inhibition of tube formation in betulinic acid treated group was 77.07 ± 9.64%. P. niruri extract against different parameters of tube formation. P. niruri in tube formation assay.Dose-dependent inhibition of differentiation of EA.hy926 cells into tube-like structures was observed in 50% ME of P. niruri extract resulted in dose-dependent inhibition of VEGF release from HCT 116 cells. Percent inhibition of VEGF released in P. niruri extract treatment groups (200 and 400 µg/mL) was 44.27% and 67.11%, respectively. As shown in Mitochondrial dysfunction and the overload of fats, along with insulin resistance, are considered the main pathological factors of NAFLD development. The accumulated fat results in endoplasmic reticulum (ER) stress, with a rise in the levels of reactive oxygen species (ROS) inside the hepatocytes in the liver . This oxMany recent studies proposed that adipose visceral tissue is an essential metabolic and inflammatory organ, and secretes an array of hormones (adipocytokines) which are transported directly to the liver though its portal vein .P.niruri treatment which also reversed steatosis as well as liver fibrosis, is a clear indicator of the therapeutic effects of P. niruri on both adipose tissues and liver.According to the current data, oral feeding with the HFD has resulted in obvious dysfunction in adipose tissue, shown by the significant reduction in the serum levels of adiponectin, with significant increase in TNF-α, IL-6, RBP4, progranulin, and vaspin levels compared with normal diet group. However, ameliorating these pathological changes by 50% ME of P. niruri improved insulin sensitivity and attenuated liver inflammation. Adiponectin, with its insulin-sensitizing and anti-inflammatory effects in NAFLD model, was one of the main factors to prevent fibrosis by reducing the concentrations of TNF-α and IL-6, and consequently, inhibit the inflammatory response. We propose that elevated circulating levels of adiponectin are likely to promote the AMPK signaling pathway in hepatocytes, suppress de novo lipogenesis by inhibiting SREBP1c, ChREBP, ACC, and FAS, as well as suppressing ER stress by activating CPT-1 to improve the β-oxidation of FFAs, leading to significant increase in insulin sensitivity with protective effects against hepatic steatosis and insulin resistance. These results are in agreement with the reported effects of adiponectin in NAFLD [Adiponectin is involved in increasing insulin sensitivity and fatty acid oxidation through the activation of PPARα and AMPK pathway. It prevents cellular proliferation and cell death by enhancing ceramide catabolism . Adiponein NAFLD . Moreovein NAFLD .P. niruri and metformin decreased the serum levels of both TNF-α and IL-6 in HFD-fed rats. This result is in agreement with previous experimental work which has reported the anti-inflammatory activity of P. niruri [Tumor necrosis factor-α (TNF-α) enhances lipolysis and fatty acid release from adipocytes, blocks insulin signals, and stimulates hepatic lipogenesis through activating c-Jun N-terminal kinase (JNK1), which also inhibits insulin signals . TNF-α p. niruri  and metf. niruri .Retinol binding protein 4 (RBP4) is one of the transport proteins for vitamin A, and its higher levels are associated with insulin resistance. It was demonstrated that RBP4 stimulates lipogenesis in hepatocytes by activating the expression and translocation of SREBP1, inducing the expression of downstream genes, such as FAS and ACC1, which all mediate the de novo lipogenesis in the hepatocytes and block insulin signals . This liP. niruri. These data confirmed the reported effect of metformin to reduce RBP4 as an indicator for an improvement of insulin sensitivity to prevent steatosis [P. niruri to reduce RBP4 as an effective pathway to prevent steatosis and reduce insulin resistance.In the current work, findings showed that serum levels of RBP4 were decreased in groups treated with metformin and teatosis . HoweverProgranulin is another adipokine linked with insulin resistance and obesity. It is usually not expressed in normal hepatocytes, but is highly expressed in the liver of NASH mouse model induced by a high-fat diet . ProgranP. niruri-treated rats was significant, while metformin treatment was able to induce considerable reduction in serum adipocytokine concentrations.Similarly to what was reported in other pharmacological studies, we have noticed, in our study, that progranulin serum levels were higher in the HFD group, confirming its positive relationship with insulin resistance and HOMA-IR, and its association with hepatic damage and fibrosis that was reported previously . The decVaspin is one of the most novel recognized adipokines, and it is known as a visceral adipose tissue (VAT)-derived serine protease inhibitor. It was proposed that vaspin serves as an insulin sensitizer with anti-inflammatory effects. Increased circulating concentration of vaspin during insulin resistance in obesity can be explained by its compensatory mechanism, targeting adipose tissues and antagonizing the disrupting effects of unknown proteases in insulin action , such asP. niruri could decrease serum vaspin concentrations compared with the HFD group, but they were significantly increased in metformin-treated rats. This may be explained as a compensatory increase, which was reported for vaspin in previous studies [Findings of our current work demonstrated that circulating vaspin concentration was increased in HFD rats compared with rats fed a normal diet. This result confirmed that elevated vaspin represents a bodily defense against NAFLD . However studies . However studies . In agre studies . As a rePPARγ is a novel member of the PPAR family which was suggested to be a major regulator in adipose tissue. PPARγ exhibits a great adipogenic action in the pathological state of NAFLD, and it stimulates the metabolism and storage of lipids . It was Our gene expression results showed that there was significant positive association between the upregulation of PPARγ and the elevated circulating levels of progranulin, vaspin, and RBP4 in the HFD group which represents NAFLD rats. This relationship was also investigated by .Collagen type 1 α1 (Collα1) gene is a tumor-related gene. This human gene has been described to be a vital component of hepatic fibrosis regulated by TGF-β in activated HSCs . It has P. niruri extract and metformin is associated with an effective reduction in the hepatic mRNA of PPARγ, SLC10A2, and Coll α1. These results provide a wealth of evidence about the beneficial effect of targeting these genes in treatment of NAFLD.SLC10A2 is a member of the sodium/bile salt cotransporter family, which has been identified in human and rats ,60. SLC1A recent study by Gao et al. has proposed that the pathological changes in circulating levels of adipocytokines during NAFLD progression are linked with the expansion of adipose tissues, concomitantly with a release of extracellular vesicles from adipocytes, which is involved in stimulating insulin resistance and augmenting growth factor β expression, inflammation, and fibrosis in the liver . MoreoveP. niruri was effective in attenuating NAFLD, preventing fibrosis, and controlling the levels of serum adipocytokines, and exhibited, at the same time, a potent antiangiogenic activity. The anti-fibrotic activity of P. niruri was associated with significant antiangiogenic effect by preventing the development of rat aorta micro vessels, diminishing the migration and differentiation of endothelial cells.The findings of the current study are in agreement with the hypothesis of both of Gao et al. and Kim et al. It was revealed that 50% ME of P. niruri of VEGF are in accordance with the previous report which suggest that blocking VEGF, the activator of HSC, is one of the mechanisms of inhibiting fibrosis [Our current data about the inhibitory effect of fibrosis .P. niruri is not related to the cytotoxic nature, but is possibly more related to blocking specific angiogenesis cascades.Furthermore, the results of MTT test with rat aortic ring analyse and the effect on EA.hy926 cells confirmed that the antiangiogenic potency verified by P. niruri extract revealed that ellagic acid and phyllanthin were major components [Standardization of current 50% mponents . Therefomponents . Ellagicmponents . In addimponents . Our resmponents  about thPhyllanthin, the other major component of the current extract, has been shown to be capable of reducing the expression of lipolytic genes in white adipose tissues of mice, and can reverse oxidative stress and inflammation in the liver and adipose tissues. It precipitates a significant reduction of liver lipids, serum triglycerides, and free fatty acids in HFD-fed mice. It was also shown to be active in terms of decreasing serum pro-inflammatory cytokines and insulin resistance . More imP. niruri extract and 500 mg/kg of metformin prevented hepatic fibrosis. P. niruri extract exhibited a comparable effect as metformin, indicating that it can be a naturally effective therapeutic choice. We also propose that the mechanism by which P. niruri elucidated this activity is multifactorial (P. niruri treatment, the abnormal alterations in all adipocytokines were rescued, and led to consequent similar improvements in insulin signaling inside the liver. This improvement resulted in a reduction of inflammation, as well as fat accumulation, and contributed in the downregulation of PPARγ, SLC10A2, and Coll α1 genes. Moreover, the beneficial effects of 50% ME of P. niruri against NAFLD progression may be also due to its potent antiangiogenic effect, which is responsible for attenuating the pathological expansion of adipose tissues and regulating its function, leading to the adjustment of adipocytokine secretion, improvement in insulin signaling, and prevention of inflammation and fibrosis in the liver. However, the limitation of the current study is that further toxicity studies of 50% ME of P. niruri are still needed, and additional studies are required to elucidate the beneficial effect of formulating P. niruri with other herbal extracts to integrate the efficacy against NAFLD.In conclusion, our results clearly demonstrated that eight weeks of HFD diet feeding in SD rats induced a significant dysfunction in adipose tissues among NAFLD rats, shown by the major reduction in adiponectin, and the considerable increase in vaspin, progranulin, RBP4, IL-6, and TNFα, leading to upregulation of PPARγ, SLC10A2, and Coll α1 genes, which are all involved in insulin resistance, hepatic steatosis, and fibrosis. Our data also revealed that oral administration of 1000 mg/kg of 50% ME of actorial , but it


Using electron microscopy and piezoresponse force microscopy, it is possible to reveal micrometre-sized wedge, lamellar-like, and irregularly shaped domains. By increasing the temperature, the domain structure persists up to 80 °C, and then starts to disappear at around 100 °C due to the proximity of the ferroelectric–paraelectric phase transition, in agreement with macroscopic dielectric measurements. In order to understand to what degree domain switching can occur in the ceramic, the mobility of the domain walls was studied at ambient temperature. The in situ poling experiment performed using piezoresponse force microscopy resulted in an almost perfectly poled area, providing evidence that all types of domains can be easily switched. By poling half an area with 20 V and the other half with −20 V, two domains separated by a straight domain wall were created, indicating that Pb(Fe1/2Nb1/2)O3 is a promising material for domain-wall engineering. A complex domain structure with variations in the morphology is observed at ambient temperature in monoclinic Pb(Fe While some compounds belonging to this class are relaxor ferroelectrics (or “relaxors” for short), such as Pb(Mg/31Nb/32)O3 and Pb(Zn/31Nb/32)O3 [33 ≈ 140 pC·N−1, a coupling factor k33 ≈ 0.27, and a saturation polarisation ≈ 11 µC·cm−2 were reported [PbO3 , PFN exhNb2/3)O3 ,7,8,9. Nreported  had beenThe domain structure was previously only investigated in PFN single crystals by optical  and TEM 3 ceramics, wedge-shaped domains were observed using electron microscopy [In polycrystalline samples, the ferroelectric domain structure was only studied in modified PFN. For Li-doped PFN, the piezoresponse force microscopy (PFM) study revealed stripe-like ferroelectric domains , but thecroscopy . In this study, we investigate the ferroelectric domains and their dynamics in polycrystalline PFN. The Rietveld refinement of the X-ray powder diffraction (XRD) shows that the samples are monoclinic. The domain structure is investigated using three complementary microscopies, i.e., PFM, TEM, and scanning electron microscopy (SEM). Each of these methods has their own limitations, but together they can describe the ferroelectric domain structure more comprehensively. The temperature evolution of the domain structure was performed with an in situ PFM experiment, revealing that the domain structure starts to disappear at around 100 °C, when crossing the Curie temperature, as determined from the dielectric permittivity vs. temperature curves. Furthermore, in this work, the emphasis is given also to the investigation of the domain-switching behaviour by applying a local DC electric field in PFM, resulting in an almost complete poling of the chosen area at ambient temperature. The local switching behaviour is supported by macroscopic measurements of polarisation versus electric field loop and the piezoelectric coefficient. 2O3 , and Nb2O5  were used. The homogenised stoichiometric mixture (200 g) was mechano-chemically activated in a planetary ball mill  for 30 h at 300 min−1 using a tungsten carbide milling vial (volume 250 cm3) and milling balls . The synthesised powder was milled in an attrition mill with yttria-stabilised zirconia (YSZ) milling balls  in isopropanol for 4 h at 800 min−1. The powder was then uniaxially pressed into disks and further consolidated by isostatic pressing at 300 MPa. The powder compacts were reactively sintered in double alumina crucibles in the presence of a PFN packing powder with the same chemical composition to avoid possible PbO losses. The compacts were sintered at 1000 °C for 2 h in an oxygen atmosphere, as previously suggested in [−1. The density of the sintered pellets determined by Archimedes’ method was ~96% of the theoretical density [For the synthesis of the PFN powder, PbO , Feested in ,15. The  density . 2 particles for 1.5 h) followed by thermal etching at 750 °C for 5 min for the thermally etched surface examination. The microstructure was examined with a field-emission scanning electron microscope  at 15 kV. For the stereological analysis, more than 340 grains were measured using the Image Tool Software [For the microstructural analysis, the PFN samples were fractured for a fracture-surface examination, ground and polished using standard metallographic techniques for the polished-surface examination, and fine-polished  and an X’Celerator detector. The XRD pattern was collected over the 2θ range of 15–120° with a step of 0.008° and an integration time of 100 s per step. In order to determine the phase structure, a Rietveld refinement analysis was performed (The X-ray powder diffraction (XRD) pattern of the crushed PFN pellet was recorded using a PANalytical X’Pert PRO  high-resolution diffractometer with Cu-Kerformed .−1, to release the internal stresses formed in the material during cutting and polishing. The piezoresponse images were recorded with an atomic force microscope  equipped with a PFM module. A tetrahedral Si tip coated with Ti/Ir  with a curvature diameter of 30 ± 10 nm was applied to scan the sample surface. The spring constant and the resonance frequency of the cantilevers were 2 N·m−1 and 70 kHz, respectively. The electric field was applied through the sample. The out-of-plane amplitude and phase PFM images were measured in the dual AC resonance-tracking mode. The used frequencies for scanning the AC electric field were 310 and 350 kHz. The local hysteresis was measured in the switching-spectroscopy mode [Prior to the PFM analysis, the samples were cut and polished using standard metallographic techniques. The samples were then heated to 600 °C for 1 h and then cooled back to room temperature with a cooling rate of 1 °C·minopy mode  with theε′ and the dielectric losses tan δ versus temperature were measured at 1, 10, and 100 kHz with a HP 4284 A Precision LCR impedance meter during cooling in the temperature range between 25 and 175 °C. The temperature-dependent polarisation (P) vs. electric field (E) measurements were made using the commercial setup Aix-PES . A bipolar sine wave with a frequency of 20 Hz was used as the input signal. The piezoelectric coefficient d33 was measured at 100 Hz of stress frequency using a standard Berlincourt piezometer (Take Control).For the electric measurements, the surfaces of ~200 μm-thick samples were covered by sputtered Cr/Au electrodes with diameters of 5 mm. The dielectric permittivity Cm, as determined by the Rietveld refinement . At ambient temperature, the tan δ is 0.038 at 1 kHz. The P–E measurement at room temperature is shown in rP) and maximum polarisation (maxP) are 16.5 and 31.1 µC·cm−2, respectively. The ɛ′, rP, and maxP are slightly higher than the previously reported values [d33 is 140 pC·N−1, which is similar to the previously reported value [The temperature dependences of d values ,22,23. TWe continue by investigating the ferroelectric domain structure. At room temperature, a complex domain configuration with variations in the morphology and a characteristic length is observed, confirming the long-range ferroelectric order, which is in agreement with the piezoelectric and ferroelectric properties. The PFM out-of-plane amplitude and phase images are shown in The first type is the wedge domains that were already observed, in both cases by TEM, in PFN polycrystalline samples  and singIn our case, the wedges appear overlapping, as can be seen in The second type is the periodic or so-called lamellar-like domains (example shown in 1/2Nb1/2)O3 and BaTiO3 [The last type of ferroelectric domains observed in our samples are the irregularly shaped domains or so-called finger-print domains  and single crystals  differ aVDN are ~9 V. The domain-switching behaviour was further studied by in situ poling, as shown in VDN of the sample. The “read” process of the locally switched area was performed by applying similar scanning conditions as prior to the application of a DC voltage. We were able to in situ pole the scanned area almost perfectly (as seen in In this study, the switching behaviour of the ferroelectric domains in the PFN material was investigated for the first time. The PFM amplitude and phase hysteresis loops are shown in P–E measurements. The results are supported by scanning transmission electron microscopy, which revealed that the samples are chemically homogeneous. The characteristic domain structure can be well correlated between the PFM, SEM, and STEM techniques, confirming that these methods are complementary. By increasing the temperature, the ferroelectric domain structure is visible up to ~80 °C, and then it starts disappearing at ~100 °C, which coincides with the dielectric permittivity peak at 98 °C. Furthermore, custom-made domain walls can be created by poling neighbouring areas with voltages of opposite signs at ambient temperature, providing a proof-of-concept for domain-wall engineering in PFN ceramics.In this study, ferroelectric domains with different morphologies and sizes—from a few hundred nanometres to micrometres—were observed by piezoresponse force microscopy. The ferroelectric domain structure is in agreement with its long-range ferroelectric behaviour, observed by macroscopic


The structural wealth of complex polyketide metabolites produced by bacteria results from intricate, highly evolved biosynthetic programs of modular assembly lines, in which the number of modules defines the size of the backbone, and the domain composition controls the degree of functionalization. We report a remarkable case where polyketide chain length and scaffold depend on the function of a single β‐keto processing domain: A ketoreductase domain represents a switch between diverging biosynthetic pathways leading either to the antifungal aureothin or to the nematicidal luteoreticulin. By a combination of heterologous expression, mutagenesis, metabolite analyses, and in vitro biotransformation we elucidate the factors governing non‐colinear polyketide assembly involving module skipping and demonstrate that a simple point mutation in type I polyketide synthase (PKS) can have a dramatic effect on the metabolic profile. This finding sheds new light on possible evolutionary scenarios and may inspire future synthetic biology approaches. Biosynthetic switch: Genetic and chemical analyses revealed a remarkable case where polyketide chain length and scaffold depend on the function of a single β‐keto processing domain of a multimodular polyketide synthase. A ketoreductase domain functions as a molecular switch between diverging pathways, leading either to the antifungal aureothin or to the nematicidal luteoreticulin. Modular type I polyketide synthases (PKSs) assemble a broad range of ecologically and pharmaceutically relevant molecules.aur, 1)syn. spectinabilin),aur PKS we have engineered an artificial pathway for the nematicide luteoreticulinsyn. griseulin,2).1, an isomeric pyrone ring, and an altered substitution pattern. Although it has been feasible to morph the aur PKS into an artificial luteoreticulin  assembly line ,To gain insight into such evolutionary processes and to emulate possible recombination scenarios we have studied in detail the biosynthesis of structurally related bacterial polyketides that share characteristic nitroaryl and pyrone moieties.Streptomyces thioluteus produces minute amounts of 2, we revisited reported luteoreticulin producers .2. Given that both polyketide metabolites (1 and 2) differ in both size and shape, it is remarkable that they seem to derive from the same modular assembly line.Prompted by the surprising observation that the aureothin producer strain 2 as a byproduct of the aur PKS  indicated that KR2 belongs to B1‐type KRs, which are characterized by a diagnostic LDD motif (VDD in KR2) and the absence of a Trp eight residues upstream of the catalytic Tyr.2, we inactivated the KR2 domain. Therefore, we replaced tyrosine with phenylalanine in the catalytic motif YAAAN by site‐directed mutagenesis of the PKS gene cloned into plasmid pHJ48 .To test whether a complete shutdown of KR2 has an impact on the production of S. albus::pHY127 and S. albus::pHJ48 were compared. A strong yellow pigmentation indicated an altered metabolite spectrum of the KR2 null mutant , whereas 1 could only be detected in trace amounts (0.36 mg L−1)  PKS (0.1 mg L−1).The metabolic profiles of S. albus::pHY140 . To obtain the ER2 null mutant, S. albus::pHY147 , we changed the conserved NADPH‐binding motif GGVGMA1 and 2, which can only be detected in trace amounts, several new compounds (3–5) are produced , 4 (24.7 mg), and 5 (3.6 mg) from 1 L culture of DH2 null mutant , and their structures were elucidated by 1D and 2D NMR analysis  impaired ketoreduction, or b) oxidation of 3, possibly by AurH. To elucidate the biogenesis of these oxygenated compounds, we heterologously produced AurH in E. coli used recombinant AurH for biotransformation experiments. In vitro assays showed that AurH transforms 3 into 4 and 5 , compound 6 and 7 , which is mediated by the methyltransferase AurI.2 are detectable in all mutants. The formation of 2 can be explained by the non‐quantitative reduction of the β‐keto group in module 2 as observed in the wild‐type PKS . Traces of 1 in the DH2 null mutant can be explained by non‐enzymatic dehydration of the hydroxy intermediate, or by the involvement of a long‐range acting DH domain as in iso‐migrastatin biosynthesis.From the HPLC profile of the ER2 null mutant, we detected two new compounds, 2 primarily depends on the dysfunction of the KR. Furthermore, it is remarkable that no module skipping takes place when the β‐keto group is processed into either β‐hydroxyl or enoyl groups. This finding indicates that the unreduced β‐keto group in module 2 is essential for the skipping process required for lut biosynthesis. Since AT4 is smaller than typical AT domains, and the function of AT4 can be substituted by AT3 from the penultimate module,The key message of these mutational experiments is, however, that the biosynthesis of 2 was still produced as the main product of the KR2 null+aAT4 mutant   or by the sequential single elongation of mutated module 2 and module 3 (lut pathway). While substrate specificities of KS domains are best studied for trans‐AT PKSs,aur KS2 domain downstream of iterative module 1 exhibits a similar gatekeeping function.To test this hypothesis, we replaced AT4 with AT3 in the KR2 null mutant. HPLC‐MS analysis revealed that Seminal studies have shown that mutagenesis and domain swaps of modular PKS lead to mainly predictable derivatives of the parent polyketide backbones.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Signal transduction through cellular membranes requires the highly specific and coordinated work of specialized proteins. Proper functioning of these proteins is provided by an interplay between them and the lipid environment. Liquid-ordered lipid domains are believed to be important players here, however, it is still unclear whether conditions for a phase separation required for lipid domain formation exist in cellular membranes. Moreover, membrane leaflets are compositionally asymmetric, that could be an obstacle for the formation of symmetric domains spanning the lipid bilayer. We theoretically show that the presence of protein in the membrane leads to the formation of a stable liquid-ordered lipid phase around it by the mechanism of protein wetting by lipids, even in the absence of conditions necessary for the global phase separation in the membrane. Moreover, we show that protein shape plays a crucial role in this process, and protein conformational rearrangement can lead to changes in the size and characteristics of surrounding lipid domains. The lipid matrix of cellular membranes is quite diverse in composition and contains hundreds of different types of lipids . FundameGenerally speaking, under the condition of constant total area of the ordered phase, the characteristic size of lipid domains in equilibrium is determined by the competition of two physical factors: entropy of the ensemble of domains and the energy of the linear boundary between ordered and disordered lipid phases. The entropy factor favors an increase in the number of clusters and, accordingly, a decrease in the size of an individual cluster. The entropic energy contribution depends only on the temperature and it is independent on physical properties of the coexisting phases. The energy of the boundary between domain and its environment contributes to the merging of domains with a decrease in their number and total perimeter, and formation of a macroscopic phase ,29. It iThere are evidences that conformational and functional states of some membrane proteins can be associated with a local change in the phase state of the lipid bilayer around them and the formation of lipid-protein domains (nanoclusters) with the size of several tens of nanometers . CertainA well-studied example of transmembrane protein distributed in a liquid-ordered phase of cellular lipid membrane is given by epidermal growth factor receptor (hereinafter—EGFR), belonging to the class of bitopic proteins, receptor tyrosine kinases . The EGFWe previously showed that formation of liquid-ordered domains around TMD of proteins can occur by the incomplete wetting mechanism . It manim being a number of components, we use Slezov model, according to which the composition of the formed ordered phase does not depend on the size of its domain [νi  parts of a lipid molecule of the i-th type, so that μeq is the chemical potential of the quasimolecule in the domain, and iμ is the chemical potential of the i-th component in the liquid-disordered phase. μeq can be related to the grand potential of the wetting ordered phase, W. It is convenient to use the grand potential in the analysis of the wetting since its independent variables are temperature and chemical potential, which are homogenous throughout the membrane in equilibrium. We denote the total number of quasimolecules in the domain as n, the average area per quasimolecule as a and the total area of the domain as s. Then, according to the well-known thermodynamic relation [To describe the wetting process, we use the Landau-Lifshitz theory . We conss domain . Formallrelation , we obtas → ∞. This normalization is chosen solely for convenience since only the difference in chemical potentials has a physical meaning, while an absolute value of the chemical potential at given conditions can be set arbitrary. The infinite increase of the ordered phase area corresponds to the conditions of a global phase separation in the membrane, thus, in a saturated membrane (with zero subsaturation) the chemical potential of quasimolecules is equal to zero. Further, we separately consider cases of the bilayer and monolayer subsaturation. In the first case, the phase separation at a given temperature does not occur in any of the monolayers, i.e., both monolayers of the membrane are subsaturated and their subsaturations are considered equal. The bilayer domain formed in this case is characterized by the total area of the ordered phase s in the outer and inner monolayers of the membrane, and radii of the domains in the outer (uR) and inner (dR) monolayers are determined by minimizing the free energy of the system. In the case of monolayer wetting, it is assumed that phase separation occurs in one (outer) monolayer of the membrane at a given temperature, i.e., this monolayer is saturated, but in the inner monolayer the global phase separation does not occur, i.e., the inner monolayer is subsaturated. In this case, the state of the system is determined by the radius of the ordered domain r, which is formed in the inner monolayer due to the wetting. Thus, in the case of bilayer subsaturation, we consider the dependence μ(W(s)), and in the case of monolayer subsaturation, we consider μ(W(r)).We normalize the chemical potential to be equal to zero for iμ can be written as follows:ic is the activity of the i-th component in the surrounding membrane, Bk is the Boltzmann constant, T is the absolute temperature. Difference between activities ic and Let us consider the right side of Equation (1). The chemical potentials μ(s) or μ(r) as a straight line parallel to the abscissa for μ < 0. The intersection point of this line with the chemical potential plot determines the domain size corresponding to the given subsaturation. The resulting domain might be stable, metastable, or unstable. In the case of several intersection points, we obtain several domain sizes corresponding to the same subsaturation. In order to determine which domain is stable, one should consider the total free energy E of the system . A stable domain corresponds to the global minimum of the free energy E, considered as a function of the domain size. This function is defined according to the expression:In accordance with Equation (4), one should define the subsaturation Δ to determine the equilibrium area of the domain. Being a constant, this value is plotted in the graph of μ and subsaturation Δ, we calculate the free energy E according to Equation (5) and find the equilibrium domain size corresponding to the given subsaturation Δ. The grand potential W of the system is determined by the membrane deformation energy arisen from the incorporation of the protein TMD, as well as the energy of deformations due to the compensation of the hydrophobic mismatch at the boundary between the domain and the surrounding membrane. Membrane deformations are considered within the framework of the Hamm–Kozlov model [n, called directors, characterizing the average orientation of lipid molecules. This field is related to some surface passing inside the monolayer. The shape of the surface is determined by the field of unit normals N to it; normals are considered to be directed towards the intermonolayer surface of the membrane. We take into account two deformation modes—tilt and bending. Deformations and elastic moduli are referred to the so-called neutral surface, on which bending and lateral stretching deformations are energetically independent. Bending deformation is quantitatively described by the divergence of the director along the neutral surface, and tilt deformation is described by the tilt vector t = n/(nN) − N ≈ n − N. We assume that membrane deformations are small. The energy of the deformed monolayer, measured from the state of a flat monolayer, can be represented as [B and K are bending and tilt moduli, respectively; σ is the lateral tension of the monolayer; dS is the neutral surface area element; A0 is the neutral surface area in the initial undeformed state. The description of deformations within the framework of such a thermodynamic approach does not allow taking into account any large-scale fluctuations , but is focused on the consideration of the membrane deformations in the vicinity of the protein inclusion, at the distances of the order of several nanometers under the assumption of their rapid decay. There are several similar models that describe elastic properties of membranes by introducing deformation modes. They include, for instance, the model proposed by Fournier [Utilizing the dependence between ov model . We intrented as :(6)W=∫BFournier . In thisFournier . This alFournier . In the Fournier ,32,33.Ozr with Oz axis coinciding with the axis of symmetry of the protein and Or axis lying in a plane perpendicular to Oz. The origin of coordinates O is located in the plane of the intermonolayer surface of the undeformed membrane. Due to the rotational symmetry, functions determining the shape and deformation of the membrane depend only on the radial coordinate, i.e., the system becomes effectively one-dimensional. In this case, the vector values of the directors, normals to the neutral surface, and tilt vectors can be replaced by their projections onto the Or axis: n → rn = n, N → rN = N, t → rt = t. Within the required accuracy, the divergence of the director along the neutral surface can be written in the form: div(n) → dn(r)/dr + n(r)/r. In addition, we take into account the condition of local volumetric incompressibility in the following form [h is the thickness of the monolayer at a given point on the neutral surface; h0 is the thickness of the unperturbed monolayer. The thickness of the unperturbed ordered monolayer is denoted by rh, and the thickness of the unperturbed monolayer of the surrounding membrane is denoted by sh. Values related to the outer and inner monolayers are marked by the indices “a” and “b”, respectively. The shape of the neutral and intermonolayer surfaces H(r) and M(r) are described by the distance from the Or plane to the point with coordinate r on the surface, measured along a perpendicular to the plane Or. In such designations the thickness of the outer monolayer is written in the form ah(r) = aH(r) − M(r), the inner one is given by the equation bh(r) = M(r) − bH(r). Within the required accuracy, the radial projection of the normal vector to the neutral surface of the outer and inner monolayers equals to aN = adH/dr and bN = −bdH/dr, respectively. One can express the functions aH(r) and bH(r) from the Equation (7) in terms of the shape of the intermonolayer surface M(r) and the radial projections of the directors of the outer and inner monolayers, a(r) and b(r). Using the simplified definitions of the tilt vectors at = a − aN = a − adH/dr, bt = b − bN = b + bdH/dr and the incompressibility condition (7), we express the tilt vectors in terms of the functions M(r), a(r), b(r) and their derivatives with respect to the coordinate r. In addition, we take into account that within the required accuracy:aH(r) and bH(r) can be expressed through M(r), a(r), b(r) and their derivatives with respect to r, using Equation (7). We substitute the obtained relations into the elastic energy functional, Equation (6), for each monolayer. The functional of total elastic energy of the membrane equals to the sum of the elastic energies of its monolayers, which depends on three functions: a(r), b(r) and M(r). To find the extremals of this functional, we vary it with respect to the functions a(r), b(r), M(r). As a result, we obtain three Euler-Lagrange differential equations. We substitute the solutions of these equations into the functional of the total elastic energy of the membrane. The expressions for the functions a(r), b(r), M(r) obtained as a result of solving the system of Euler-Lagrange differential equations contain indefinite coefficients, which are determined from the boundary conditions and from the condition of minimum of the total elastic energy. The boundary conditions are determined by the geometry of the TMD of the protein, the requirement of continuity of directors and neutral surfaces, and the requirement of the finiteness of the deformations. The model is described in details in We assume that the protein has a cylindrical symmetry. This allows introducing a cylindrical coordinate system ing form :(7)h=h0M(r) is the same for the incompressibility conditions written for the upper and for the lower monolayers, and this imposes ideal balance of forces and torques induced by the monolayers at the intermonolayer surface. From the other hand, the membrane deformations are coupled to the composition of the leaflets via such elastic characteristics as the monolayer equilibrium thickness or elastic moduli. Thus, yet our model does not explicitly include leaflet deformations and composition coupling, it is accounted implicitly via volumetric incompressibility conditions and dependence of the elastic parameters on the lipid composition.Our approach based on the Hamm-Kozlov model of lipid membranes elasticity  does notB = 10 BTk0 , K = 40 mN/m = 10 BTk0/nm2 [rh = 1.8 nm, the monolayer thickness of the surrounding membrane equals to sh = 1.3 nm. The lateral tension of the monolayer is taken equal to σ = 0.01 BTk0/nm2, which is of the same order of magnitude as it is determined for cell plasma membranes [For the quantitative calculations, we use the elastic moduli relevant for the model lipids. Their values in accordance with experimental and theoretical data are taken equal to kBT0/nm2 ,53 for membranes . SubsatupR, the radii of ordered domains in the upper and lower monolayers, uR and dR, respectively, as well as the radial projections of the boundary directors in the upper and lower monolayers, n1 and n2, which can be different. Schematically, this model is presented in We consider the formation of a liquid-ordered lipid domain around a transmembrane protein by the mechanism of wetting. For the sake of generality, we assume that the structure of the lipid domain and the transmembrane domain of the protein does not have mirror symmetry with respect to the membrane midplane . For defpR, uR, dR, n1, n2. The obtained results are compared with the case of the epidermal growth factor receptor, for which it has been experimentally shown that a change in the conformation and, correspondingly, in the shape of TMDs of its dimer plays a key role in the activation of this receptor [pR increases from approximately 0.9 nm to 1.6 nm [n1 and n2. We assume that in the closed state of the receptor n1 < 0; n2 ≈ 0, and in the open state n1 > 0; n2 < 0 , we find the total domain area in the outer and inner monolayers eqs corresponding to the minimum of the free energy, i.e., equilibrium state of the system. Then we calculate the dependence of the elastic energy W on the radius of the domain dR in the lower monolayer for the fixed total area of the ordered phase around the protein eqs and find the value of the radius dR corresponding to the minimum of the energy W. The radius of the ordered domain in the upper monolayer uR is determined from the relation:eq s = π(dR2 − pR2) + π(uR2 − pR2). Thus, we determine the size of the domains of the liquid-ordered phase in both monolayers for each given shape of the protein TMD. In this case, a stable ordered domain can be either bilayer  or monolayer .We vary the radial projections of the boundary directors pR = 0.9 nm, n1 < 0; n2 ≈ 0), stable domains do not form. Moreover, at pR = 0.9 nm, stable domains are not formed for any physically reasonable values of the radial projections of the boundary directors n1, n2. At pR = 1.6 nm, a stable domain can form around the protein. The domain can be either bilayer or monolayer depending on the specific shape of TMD, i.e., on the value of the radial projections of the boundary directors. However, the range of values , at which stable ordered domains are formed, does not intersect with the region  corresponding to the open EGFR conformation, which is illustrated in We found that in the case of the closed conformation , we determined the ranges of these parameters in which the formation of a stable ordered domain in the lower monolayer is possible for open and closed conformations of the TMD of EGFR. The radius of the ordered domain in the outer monolayer was considered equal to uR = pR + 7 nm. The calculation results are presented in By the direct enumeration of various values of the radial projections of the boundary directors  under which the formation of a stable ordered domain in the inner monolayer is possible  , a stable bilayer domain does not form , or the spontaneous curvature of the monolayers of the membrane , nor the membrane fluctuations. The effect of hydrophobic mismatch on the possibility of the formation of stable domains was previously described in  and is tIn summary, in the present work, we developed a model describing the formation of liquid-ordered domain around transmembrane proteins in subsaturated membrane, i.e., in the absence of conditions for the global phase separation. As the driving force of the local phase separation, we considered a hydrophobic mismatch between the length of the protein transmembrane domain and the thickness of the liquid-disordered bilayer. For relatively long TMD it was energetically favorable to surround the protein by the liquid-ordered domain. The thickness of the ordered bilayer is larger than that of the surrounding disordered membrane. Thus, in the process of wetting in such system, the hydrophobic mismatch between undeformable (infinitely rigid) protein and the disordered membrane is effectively transformed to the thickness mismatch between deformable  ordered and disordered lipid phases.It was shown that the formation of stable liquid-ordered domain depends on the TMD radius. Generally, larger radius of TMD promotes wetting. Besides, the wetting effectiveness is strongly dependent on the TMD shape, which is parameterized by radial projections of boundary directors. Generally, the wetting is most effective when the projections of the boundary directors of the outer and inner monolayers are both positive , i.e., w


Modular polyketide synthases (mPKSs) build functionalized polymeric chains, some of which have become blockbuster therapeutics. Organized into repeating clusters (modules) of independently-folding domains, these assembly-line-like megasynthases can be engineered by introducing non-native components. However, poor introduction points and incompatible domain combinations can cause both unintended products and dramatically reduced activity. This limits the engineering and combinatorial potential of mPKSs, precluding access to further potential therapeutics. Different regions on a given mPKS domain determine how it interacts both with its substrate and with other domains. Within the assembly line, these interactions are crucial to the proper ordering of reactions and efficient polyketide construction. Achieving control over these domain functions, through precision engineering at key regions, would greatly expand our catalogue of accessible polyketide products. Canonical mPKS domains, given that they are among the most well-characterized, are excellent candidates for such fine-tuning. The current minireview summarizes recent advances in the mechanistic understanding and subsequent precision engineering of canonical mPKS domains, focusing largely on developments in the past year. Antibiotic resistance is on the rise, and it is essential to maintain a steady pipeline of antimicrobial candidates . Microbitrans-AT PKSs. Iterative PKSs represent a related but non-assembly-line PKS class [mPKS domain engineering has become one focus among many in the rapidly expanding field of PKS research . RelatedKS class ,10,11, vKS class ,13. BesiKS class ,15, and KS class ,17,18. RKS class . The evoKS class ,21.New assays, combined with modeling and structural visualization are enabling an increasingly detailed picture of mPKS function ,23,24,25Type 1 mPKSs build functionalized carbon chains that can further react to form macrocycles and heterocycles. These megasynthases are composed of many catalytically active domains arranged in modular clusters (modules), with each independently-folding domain housing a catalytic center for a discrete enzymatic reaction . ModulesWhile some type 1 mPKS modules have been found to ‘stutter’—catalyzing two or three rounds of chain elongation ,40,41,42While this description provides a generalized map of PKS architecture and function, it is admittedly oversimplified. It nonetheless formed the basis for the first generation of mPKS engineering, which envisioned mPKSs as having potentially ‘lego-like’ modularity . Such anIt is now clearer that the contributions of the various domains to their canonical chemical outcomes are one of the many functions—chemical, structural and logistical—that they play. For example, a DH domain may be responsible for catalyzing a dehydration reaction within a given module, but it can only work on stoichiometric amounts of substrates that have been reduced to the correct stereochemistry by an appropriate KR domain , and perOverall, multiple chemical, structural and logistical functions influencing a single chemical outcome are performed by different regions within various architecturally discrete domains. From an engineering standpoint, it is critically important to decipher which of these functions play the most predominant roles for a given chemical outcome, and assess the degree to which they can be tuned independently from their parent domain’s other functions. To this end, several engineering approaches are being honed to alter one function while minimizing disruption to other others within same domain and module, including active site ‘surgeries’ and more refined domain swaps. Key lessons from recent work will be underlined as ‘take-home messages’ for the prospective mPKS engineer.Recent cryo-EM, SAXS, and crystal structure-based visualizations of whole-module and multimodular PKS architectures ,51,52,53More evidence has been recently been published to support the hypothesis of a turnstile mechanism, namely, the single substrate occupancy of PKS modules . Using aIt should be noted, however, that there remain ambiguities in the cryo-EM structures—while they were prepared to mimic various reaction steps in an elongation cycle, the conditions weren’t replicated with complete correspondence—particularly, extra or absent acyl substrates could have plausibly had unnatural impacts on module conformation . Moreoven-KSn+1 approach for combining modules [Given the strong role of ACP-KS interactions in this turnstile mechanism, as well as in the activity outcomes of module-swapped PKS chimeras ,59,60, tOne intriguing behavior observed in the cryo-EM models was the role of the acyl chain in influencing the interaction between the upstream ACP and its KS partner. Without an acyl chain, the ACP would not even contact the KS, while its presence enabled sufficient affinity. Questions raised about the role of the substrate in ACP-KS interactions—and ACP-partner interactions, more generally—are being further investigated. Recently, NMR studies tracking the interaction of ACPs with their acyl substrates have shown that larger chains interact extensively with their carrier protein, possibly nestling within a hydrophobic pocket while leaving the thioester component displayed. This could position the thioester β-carbon for easy access and recognition by partner domains, enabling their reduction state to tip ACP affinity towards the appropriate partner .cis-AT) mPKSs are known to have relaxed substrate specificity [Cis-AT mPKS KS domains have been relatively underexplored with respect to engineering and active site residue characterization, possibly because of its involvement in so many different functions. To narrow down the selection of active site residues to explore, Robbins et al. compared 90% consensus sequences from 226 non-iterative PKSs with that of 24 iterative ones [While KSs of canonical  and dextro (d) respectively. A1-KRs accept substrates bearing a d-α-branch, though the A2-type may accept an α-branch in either configuration but will epimerize it to the L isomer. Type-B KRs can be categorized analogously  arm holding the acyl chain was implicated . Beyond This hypothesis has been supported by the results of another study, one focused on improving outcomes for the RLS approach. Eng et al. noticed that a functional sequence discrete from the KR domain  was usuThese advances in re-engineering the stereochemical outcomes of the KR domain has been complemented by investigations into its ACP specificity. The interaction landscape of four reducing KRs from DEBS and five ACPs from DEBS was assessed in a simple in vitro assay . The outtrans or cis product . Trans double bonds typically result from B-type ketoreduction, while cis double bonds are sometimes formed from A-type products [There has been comparatively less progress recently in honing control over dehydratases. Most research on these domains currently proceeds at the level of structure, substrate specificity, protein-protein interactions and reaction outcomes ,79. The products . While sproducts . Reversaproducts . Dehydraproducts .S) to (2R) [There have been few recent developments in mPKS ER engineering. Besides a well-known Tyr-to-Val mutation, which alters methyl branch orientation from (2 to (2R) , there h to (2R) . BecauseThioesterase sequences vary as much by phylum as they do by PKS family type, and factors determining their two typical choices for substrate offloading, hydrolysis or macrocyclization, remain unpredictable by sequence alone . AlthougIn the absence of clear crystal structures, detailed theoretical approaches have been undertaken to model both cyclization and hydrolysis reactions catalyzed by DEBS TE. Chen et al. used QM and MM models to investigate determinants of the reaction fate of three DEBS-like SNAC substrates . Their wA n unusually methodical domain swapping approach has been demonstrated in recent work by Hagen et al., in their development of an adipic acid-producing monomodular PKS . This chSince the RLS method has been the most successful in exchanging or adding reducing domains, reducing loops from four alternative modules were tried using two different junctions, giving a total of 8 chimeric modules. These chimeras were evaluated in vitro for their ability to elongate succinyl-CoA with a malonyl extender unit. Using a mass-spectrometry-based phosphopantetheine ejection assay , which eThe TE domain was added following the aforementioned troubleshooting and refinement, and the following in vitro assays revealed the final chimera to produce adipic acid at concentrations comparable to the amount of 3-hydroxyl-adipic acid produced by wild-type BORS M1-TE. This work demonstrates a remarkably methodical approach to PKS engineering through domain swaps, testing panels of parts, evaluating multiple junctions for each exchange, and evaluating performance of each domain through detection of reaction intermediates. We anticipate widespread use of this method in the years ahead, although its in vitro nature likely precludes high-throughput application in the short term.Significant progress has been made in mapping and fine-tuning the functions of various canonical PKS domains. The presence of a turnstile mechanism shows that the fusion points of any domain swaps should be chosen judiciously, and several options should be attempted. The KS plays a key role by interfacing with the translocating ACP to receive its acyl substrate. If the interfaces are incompatible, the loss in activity is quite significant, while incremental modifications to the substrate had a smaller effect on productivity. Within the KS, catalytic and supporting residues have been mapped and we now know that a mutation in the active-site proximal dimerization loop greatly enhances activity and substrate tolerance. ATs have been extensively engineered to enable incorporation of non-native building blocks, but often through inefficient means: domain swaps have typically caused disrupted protein interfaces, and SDM methods achieved promiscuity only by destroying natural substrate affinity. However, recent work honing both these methods have resulted in mutants and chimeras with swapped instead of damaged activity. Key determinants like modular context and binding site size have been shown to influence KR outcomes, and an overlooked role of the dimerization element in reducing loop swap outcomes has been demonstrated. While the remaining domains have seen more characterization and less engineering, TE theoretical studies have developed insightful models that provide hypothesis for further verification. Finally, an engineering tour-de-force has demonstrated how mPKS modification can be undertaken in a stepwise, methodical fashion to produce high-activity chimeras with intended catalytic ability. These advances illustrate valuable engineering principles for modular polyketide synthase engineering, and will likely enable more extensive, predictable, and independently tunable modifications.