This review focusses on the development of thermoelectric composites made of oxide or conventional inorganic materials, and polymers, with specific emphasis on those containing oxides. Discussion of the current state-of-the-art thermoelectric materials, including the individual constituent materials, i.e., conventional materials, oxides and polymers, is firstly presented to provide the reader with a comparison of the top-performing thermoelectric materials. Then, individual materials used in the inorganic/polymer composites are discussed to provide a comparison of the performance of the composites themselves. Finally, the addition of carbon-based compounds is discussed as a route to improving the thermoelectric performance. For each topic discussed, key thermoelectric properties are tabulated and comparative figures are presented for a wide array of materials. Current global concerns about energy resources see a move towards sustainable energy generation technologies with improved efficiency and performance, due to a growth in population, a greater demand for electric transportation and machinery, and stringent policies to reduce the environmental impact of anthropogenic activities. To meet such increasing energy demands while maintaining a carbon neutral target, replacing more impactful energy sources with greener solutions remains imperative.In 2020, the UK total electricity demand approached ~324 TWh with an estimated growth of ~76 TWh predicted by 2040 . AlthougFor most energy generation technologies, such as thermal power plants and photovoltaics, a substantial portion of energy loss is in the form of heat, leading to lower overall efficiency. In photovoltaics, where the exposure to sunlight is associated with thermal energy, thermoelectric (TE) devices can be implemented to recover the waste heat, converting it directly to electricity . For morA TE device takes the form of a series of thermopiles , i.e., tTo date, the promising use of TE devices is undermined by their low performance and relatively high cost. The overall efficiency of a TE device, nd 6–10% . Z is exTo improve the overall efficiency of a TE device, the performance of TE materials is crucial and must be considered on an individual basis. This is measured by the dimensionless figure of merit in Equation (3).The Seebeck coefficient, The carrier concentration is also related to the electrical conductivity, which represents the ease at which the charge carriers are conducted through the material, Equation (6).19–1021 cm−3 [021 cm−3 .S are related to the charge carrier concentration ) . Hence, A change in 2Te3 and PbTe, and can offer a respectable ZT ≈ 1.1 at room temperature (RT) [Conventional TE materials are crystalline chalcogenides in their bulk form, such as those based on Biure (RT) . Howeverure (RT) . On the ure (RT) ,21,22.ZT = 0.65 at 1247 K) [xCoO2−δ (ZT = 1.2 at 880 K) [3 (ZT = 0.20 at 1045 K) [3 (ZT = 0.37 at 1000 K) [3 (300–1100 K) [2O3 (300–1100 K) [3 (300–1000 K) [There is mounting evidence that shows competitive performance between oxide and conventional TE materials, with some of the most promising oxides being ZnO  1247 K) , NaxCoO27 K , Nax 1045 K) , and CaM 1000 K) , Due to  1000 K) , the the–1100 K) ,27, In2O–1100 K) , and CaM–1000 K) . Howevereratures ,22. At h3–723 K) . Indeed,2 µW m−1 K−2), it is implied that improvements can be made to the ZT of a material, if S and σ are maintained. Material engineering can be attempted by combining different strategies that tend to act on specific TE properties. Nanostructuring aims to introduce grain boundaries and interfaces to reduce thermal conductivity. Nanocompositing aims to mainly reduce thermal conductivity while increasing electrical conductivity by coupling two unalike materials. The adjustment of a TE material’s stoichiometry via the introduction of intrinsic or extrinsic defects is generally named band engineering and mostly aims to increase electrical conductivity and enhance the Seebeck coefficient. Of course, none of these engineering strategies is fully independent with respect to their effect on TE properties. For instance, where there are reasonable PFs  may influence the underlying physics. This is a current drawback and hinders the full exploitation of the advantages of TE composites. For oxides, perhaps the most promising strategies to enhance  species . This ree window . Recentlformance ,33. AlthZT values so that a benchmark against the best performing single materials can also be provided. In this review, we cover the composite TE materials made of an inorganic material and a polymer. The term “inorganic (TE) material” covers both oxide and conventional TE materials and is used when discussing both of them. However, when considering specific materials, whether oxides or conventional materials, these terms will be specifically used. As the names suggest, oxides are inorganic materials that contain oxygen, whereas conventional do not. The review is divided into Sections that summarise the inorganic  thermoelectric materials, and their carbon composites , polymerHere, we provide a brief overview of conventional and oxide TE materials by highlighting the current front-runners , before While we leave detailed discussions on the potential of oxide and conventional TE materials to several comprehensive reviews ,22,36,37xCoO2−δ has the highest ZT of 1.20 at 800 K. It is clear that oxides have much lower overall performance, with ZT values in the range of 0.06–1.2 compared to 0.92–2.80 of conventional TEs. It is also apparent that n-type oxide materials (0.06–0.65) suffer from moderate ZT values in comparison to p-type oxide materials (0.29–1.20). Within ZT are reported for conventional p-type TEs (GeTe)0.95(Sb2Te3)0.05 (ZT = 2.70 at 720 K) and single-crystal SnSe (ZT = 2.80 at 773 K). However, most conventional TEs operate at lower temperatures, ~RT, while oxides reach their optimal performance at higher temperatures, >800 K. A direct comparison cannot thus be made between the two classes of materials, instead here we only highlight the maximum achievable performance of the materials, irrespective of the temperature at which this is achieved. For oxides, the p-type Na0.99Sb0.01O2, Ca0.9Dy0.1MnO3 and In1.88V0.12O3) report slightly lower values. For n-type and p-type oxides, the thermopower range is 140–300 µV K−1 with an average value of ~217 µV K−1. For oxides, the highest S values are generally reported for p-type cobaltates, e.g., Bi2Sr3Co2Oy (300 µV K−1) and Ca2.97Sr0.03Co4O9 (270 µV K−1), most likely due to the characteristic degeneracy of the 3d orbitals and low-spin state of Co 3+ [0.1Eu0.9TiO3−δ, which exhibits a large thermopower of −300 µV K−1 due to the Eu2+ 4f bands located towards the top of the valence band [S [S = 190–476 µV K−1. The highest performing conventional materials have a larger thermopower than all reported oxides, e.g., SnSe (~−476 µV K−1) and Sn0.97Re0.03Se0.93Cl0.02 (~−430 µV K−1).When comparing the thermopower of oxides and conventional materials, there is an overall similarity in performance, although some oxides , Zn0.97Al0.02Ga0.02O (~570 S cm−1), In1.88V0.12O3 (385.5 S cm−1) and TiO1.76 (350 S cm−1), show that they outperform some of the best conventional materials. Indeed, the highest reported xCoO2−δ (1923 S cm−1) far exceeds that of the highest 0.08La0.05Yb0.08Co4Sb12 (1344 S cm−1). For both conventional and oxide TEs, smaller For the electrical conductivity, carriers . −1 K−2, while conventional TEs report PF in the range of 570–5269 µW m−1 K−2. For some of the most promising oxides, e.g., Zn0.97Al0.02Ga0.02O (~2309 µW m−1 K−2 at 1247 K) and Ca2.97Sr0.03Co4O9 (1200 µW m−1 K−2 at 1073 K), larger PFs are observed at high temperatures. It is seen that the highest PFs in oxides are reported almost exclusively for the p-type layered and misfit cobaltates, with the exception of the n-type zinc oxide, Zn0.97Al0.02Ga0.02O. It is also apparent that some of the perovskite-type structures perform particularly well, e.g., La0.08Sr0.92TiO3 (~1130 µW m−1 K−2 at 1045 K). The oxide with the highest PF is NaxCoO2−δ (7690 µW m−1 K−2), which is reported at a much higher temperature (800 K) compared to the several high-performing conventional TEs 0.95(Sb2Te3)0.05 and (Hf0.5Zr0.5)0.7Ti0.3NiSn0.998Sb0.002). It is also the case that the highest PF reported for conventional materials is the cobalt containing Ba0.08La0.05Yb0.08Co4Sb12, where PF = ~5269 µW m−1 K−2 at 850 K. When comparing TEs in terms of power factor, PF  or have bonding which is less conducive for phonon-transport, resulting in a low Thermal conductivity, ng modes . It is tbaltates ,71, whicposition ,73.ZT with nanostructuring/band engineering, when they are used in composites, the materials are usually not fully optimised. Furthermore, for many of the inorganic/polymer composites discussed in ZTmax for conventional and oxide TE materials used within inorganic/polymer composites. ZT, PF, σ, S and Here, the performance of oxides and conventional TE materials typically used within inorganic/polymer composites are discussed, irrespective of their n-type and p-type behaviour. Although we recognise that some of these materials could show a higher S values are reserved for oxides  and Ba0.1Eu0.9TiO3–δ  are relatively impressive, outperforming the conventional material Pb0.995Sb0.005Te (~−280 µV K−1 at 600 K), which can be classified as a competitor due to its performance in proximity of the operating temperature of oxides (>800 K). S has many dependences on both charge carrier concentration  and (5)) is dependent upon the parameters The most impressive r oxides , 100–340aterials ,80. TempS for inorganic TEs. For some oxide and conventional TEs, there is very little change in S with increasing temperature, e.g., Bi2Te3 , Ba0.1Eu0.9TiO3–δ  and Ni0.94Li0.06O , whereas for others there is a more significant change, e.g., Zn0.998Al0.02O , and Pb0.995Sb0.005Te . −1 except for Zn0.998Al0.02O (~630 S cm−1), which like its state-of-the-art counterpart Zn0.97Al0.02Ga0.02O (−1). Oxides show poor electrical conductivity compared to conventional materials, which display −1, with the highest value of 1000 S cm−1 for Pb0.995Sb0.005Te at 300 K, followed by 608 S cm−1 for nanostructured Bi2Te3. 0.1Eu0.9TiO3–δ and Ni0.94Li0.06O, it remains fairly constant in Ca2.93Sr0.08Co4O9, and decreases with temperature for Zn0.998Al0.02O. Perovskite La0.067Sr0.993TiO3 shows a more complicated relationship where a reasonable conductivity (~760 S cm−1) is shown at T > 800 K, after a sharp rise in −1). The electrical conductivity of oxides shown in 2Ga0.02O , has a hConventional TEs have a maximum attering . When a −1 K−2) than oxides, which usually sit within 100–600 µW m−1 K−2. However, the oxide Zn0.998Al0.02O (~1370 µW m−1 K−2 at 1247 K) shows a comparable performance to conventional materials and indeed exceeds the performance of Bi2Te3 (~934 µW m−1 K−2 at 325 K) irrespective of the operating temperature. As discussed previously  than conventional TE materials (0.31–0.88). Nevertheless, a few promising oxides La0.1Sr0.9TiO3 (~0.27 at 1050 K), Ba0.1Eu0.9TiO3–δ (~0.24 at 1120 K) and Zn0.998Al0.02O (~0.37 at 1247 K) display ZTmax values similar to the lowest reported conventional material Pb0.995Sb0.005Te (0.32 at 300 K). This suggests that a number of oxides show a similar performance compared to conventional TEs, and hence could yield similar or better performance at their individual optimal temperature ranges. Oxides used in inorganic/polymer composites  express −1 K−2, their lower ZT is mainly resultant from the larger −1 K−1 compared with conventional materials (0.5–2.4 W m−1 K−1). Astonishingly, at RT, NaxCoO2−δ has a low ZT = 0.036, which is attributed to a large −1 K−1 due to its layered structure [As the PFs of oxides are typically ⪆200 µW mtructure ,82.ZT values increase over the temperature range, ZT need maintaining. For numerous oxides shown in ZT values are limited to high temperatures (>800 K) with very low values reported under ambient conditions. For most oxides, ZT is achieved, allowing for possible lower-temperature applications (RT) [The addition of carbon allotropes to inorganic TE materials is a relatively new area of research with promising results obtained thus far. The manufacturing of carbon composites has been shown to broaden the operating temperature range and provide a lower temperature at which maximum ons (RT) . We makeons (RT) ,85,86,87ons (RT) ,89,90. Fons (RT) ,92,93,94ZTmax.Within this Section, examples of inorganic/carbon composites are discussed, listing some high performing composites and the rationale behind their applicability is given. A comparison to oxides and conventional TEs is made. The creation of composites usually involves the addition of organics and/or more recently polymers to a doped inorganic material, which is either in bulk or nanoparticle forms. 3 [0.067Sr0.933TiO3 generated a −1, which is greater than that of the inorganic single crystal (−1), leading to a PF = ~2400 µW m−1 K−2 at ~312 K, which is marginally larger than the inorganic single crystal (PF = ~2370 µW m−1 K−2 at ~348 K) [κ, an increase in σ, while moderate S values were maintained [0.067Sr0.933TiO3, a high PF = ~2500 μW m−1 K−2 was achieved, leading to ZT = 0.42 at RT, which is an approximately 280% increase compared to inorganic La0.067Sr0.9TiO3, and slightly higher than that of Gr (1 wt. %)/La0.067Sr0.9TiO3. This could be attributed in part to the large reduction in 0.067Sr0.9TiO3 at 315 K due to the scattering of phonons at the interfaces. The differences in TE parameters between varied loadings of graphene suggest that there is an optimal loading window for the addition of graphene, too much or too little can hinder performance. The TE parameters of the aforementioned composites are comparable at RT to those typically reported for conventional materials  has opened up to lower temperatures, in line with conventional TEs such as Bi2Te3 and PbTe. This change was also observed upon the addition of reduced graphene oxide (RGO) to undoped SrTiO3 [σ is due to mitigation of electrical impedance induced by trapped electrons at the grain boundaries. The addition of RGO to SrTiO3 leads to formation of strontium /SrTi0.85Nb0.15O3, ZT = 1.42 at 1047 K, which is a considerable increase compared to the highest performing doped SrTiO3, e.g., La0.08Sr0.92TiO3  and Gr (1.0 wt. %)/Sr0.8La0.067Ti0.8Nb0.2Oδ3- . This is perhaps attributed to the large σ = 1193 S cm−1 and a reasonable S = −187 µV K−1, as in this case −1 K−1, which is similar to that reported in oxide inorganic, La0.08Sr0.92TiO3 , suggesting that the high ZT value cannot be attributed to the slight reduction in 0.067Sr0.9TiO3 . This may be attributed to the slight decrease in −1 K−1 due to the graphene–Bi2Te3 interface and the increase in −1 brought on by the intrinsically high S = ~19 µV K−1. The performance of this composite is also closely followed by Graphene (0.6 wt. %)/La0.067Sr0.9TiO3 and CNT (0.05 wt. %)/PbTe. For conventional/carbon composite, graphene (1.5 wt. %)/BiS, σ is increased. Similar results are expected for the manufacturing of inorganic/polymer composites. As discussed within this Section, the creation of composites with carbon can lead to beneficial changes to key thermoelectric parameters. Indeed, although at the expense of −6–10−11 S cm−1), and usually have a relatively wide bandgap of 2–3 eV [Polymer TEs obey the same fundamental principles as inorganic TEs (Equations (1)–(7)). However, thermal and electronic transport may require more complex considerations; we refer the reader to the review of Xu et al. . Most pof 2–3 eV . Chemica polymer . The inccription ,110. Othcription , whereascription . Whilst the n-type polymers discussed in this Section cannot be easily split in to general classes, the p-type polymers can be split into two classes: those containing sulphur, i.e., polythiophenes  and those containing nitrogen. The latter can be further divided into two categories: nitrogen within the aromatic system , and nitrogen outside the aromatic system . Here, we begin by introducing the current front-runners in polymer TE materials  to highlZTmax for the highest performing polymer thermoelectrics within the current literature. We leave detailed discussion of the potential of polymers for TE applications to several comprehensive reviews [ reviews ,114,115.−1). When −1  TEs. The polymer PEDOT:PSS/EMIM-DCA also exceeds the highest reported 0.998Al0.02O, ~630 S cm−1, −1), which express much lower −1).While all the TE polymers in 0 S cm−1 , which dcm−1 −1,  and oxid −1, −1,  TEs. Theon (6)). . When co−1. This makes them competitive compared with some of the top performing inorganic materials, e.g., Pb0.92Mg0.08Se0.2Te0.8  and conventional (190–476 µV K−1) TEs (−1) shows great promise, with a magnitude of S similar to a number of the highest performing inorganic TEs. It is important to remember that both S andS. This can be explained by the relationship between S and S. Conversely, the use of various dopants may alter the effective mass of the carrier S. When we consider the different polymer classes, the N polymer, PANI (~20 µV K−1) exhibits the lowest S value, which in performance is similar to the lowest reported PTh polymer, PBTTT/FTS (33 µV K−1).p-type polymers outperform n-type polymers in terms of K−1) TEs . HoweverZT. The PF of p-type polymers is in the range of 11–1270 µW m−1 K−2, while the ranges of PFs for conventional and oxide TEs are 578–5269 µW m−1 K−2 and 359–7690 µW m−1 K−2, respectively (−1 K−2 compared to the p-type polymers (0.067Sr0.993TiO3 (~416 µW m−1 K−2) and Ca2.93Sr0.08Co4O9 (~298 µW m−1 K−2). It is clear that most polymers do not compete with conventional TEs at RT, e.g., Pb0.995Sb0.005Te (~2410 µW m−1 K−2 at 325 K), apart from the front-runners acid-base treated PEDOT:PSS/EMIM-DCA (754 µW m−1 K−2) and PP-PEDOT:Tos (1270 µW m−1 K−2 at 300 K). Additionally, when comparing between polymer classes, the N polymer, PANI (~11 µW m−1 K−2) has the lowest PF of the p-type polymers, far less than the lowest reported for the PTh class, e.g., P3HT (~46 µW m−1 K−2). Many papers investigating TE properties of polymers report only values for PF instead of ectively . The n-tpolymers  that are−1 K−1, −1 K−1) and oxide TEs (0.60–7.94 W m−1 K−1) over their operating ranges  and conventional  TEs, which operate around RT, polymers show much lower −1 K−1 at 300 K and PEDOT:PSS, 0.52 W m−1 K−1 at 300K) [ZT  and Bi2Te3 (~0.25) at ~RT  ,82. This) at ~RT . The oveZTmax or at the maximum of PF, for those polymers used in inorganic/polymer composites. ZT and Conductive polymers, such as poly (PEDOT), polyacetylene (PA), polyaniline (PANI), polypyrrole (PPy) and polythiophene (PTh) derivatives, are p-type thermoelectrics when oxidised . Due to The two classes of polymers have different reported operating temperature windows: 300–450 K for N and NAr, and lower temperatures 150–300 K for the PTh polymer, PEDOT:PSS . −1, −1, −1 comparable to the highest reported oxide TE, NaxCoO2−δ  are reported in the range 11–169 µV K−1  and Pb0.995Sb0.005Te (~−157 µV K−1 at 300 K)  . −1 K−2 ; however, this is still far lower than the ranges of PF values for both oxide (359–7690 µW m−1 K−2) and conventional TEs (578–5269 µW m−1 K−2). The power factors of polymers are situated in the range 0.08–141 µW m−1 K−2  with val−1 K−1. These values are inherently low when compared to the −1 K−1) and conventional (0.22–3.10 W m−1 K−1) TEs, which tend to be much higher  and show an overall increase in ZT with increasing temperature. The  general . For ZT S resulting in higher PFs. A number of reviews have been published on polymer/carbon composites [σ, S, The manufacturing of polymer/carbon composites TEs is much more commonly explored than that of inorganic/polymer composites. As we leave their detailed discussion to a number of reviews ,137,138,mposites ,128,134;−3–1900 S cm−1, with most common values in the 102 S cm−1 range. This is a substantial improvement when compared to polymers alone  and nanostructured Bi2Te3 (~610 S cm−1). The 0.067Sr0.933TiO3 (1780 S cm−1). Meanwhile, a significant number of polymer/carbon composites have a very impressive 3 S cm−1 with some of them with a far greater 0.08La0.05Yb0.08Co4Sb12 (~1344 S cm−1), and similar to/exceeding the highest reported xCoO2−δ (~1923 S cm−1). For example, the PANI/Gr-PEDOT:PSS/PANI/DWNT/PEDOT:PSS (1900 S cm−1) and PANI (80 wt. %)/GO (1500 S cm−1) exhibit a far greater 0.08La0.05Yb0.08Co4Sb12, with the former composite having a similar xCoO2−δ. For −1 at 80% polymer loading, while PEDOT:PSS/Gr shows maximum, −1 at 20% polymer loading.Many anic TEs , e.g., Zmposites , with soS is positive for all but one composite (PVAc (20 wt. %)/GTNC), suggesting that p-type conductivity dominates polymer/carbon composites. Overall, the reported S values are rather low with typical values around S = 101 µV K−1. The reported range of S is 6–130 µV K−1 with the highest thermopower belonging to the PANI/Gr/PANI/DWNT composite (130 µV K−1), which is competitive with some conventional TEs at RT, e.g., Pb0.995Sb0.005Te (−154.24 µV K−1). When comparing S of polymer/carbon composites to typical polymers , PANI/Gr/PANI/DWNT (1825 µW m−1 K−2), PANI (80 wt. %)/GO (~521 µW m−1 K−2), as these compare to several inorganic TEs  and Graphene-oxide/PANI (80 wt. %) composites. On addition of carbon fillers, similar or slightly larger ts alone . Howeverat 420 K  while Gr40 at RT ,141. Thianic TEs , namely,ZT must be >1. However, lower power applications may still be applicable such as “wearable” technologies [S, S and The TE parameters of polymer/carbon composites do not compare to those of inorganic TEs . For an nologies ,128. PolThe nanoscale structure is fundamental to TE performance. Research focusing on structural features such as defect clusters, voids, and interfaces often yields promising results in oxide and conventional TEs ,162. TheS and σ. As the value of S is mainly determined by the energy of the Fermi level and the position of the band edge [S and σ. The decrease in σ can further be explained as a reduction in σ. In theory, the band structure of a composite may be improved by the introduction of a higher abundance of highly conductive fillers such as graphene, polymers etc.One of the challenges of creating composite materials is maintaining or improving both and edge , the forion (6)) ,167. An Here, we present inorganic/polymer composites which are relevant to TE applications. Within The combination of conventional TE materials and polymers will be briefly discussed along with a comparison made to both the individual polymer and the conventional TEs. For an in-depth discussion of the topic, we refer the reader to existing reviews ,168,169.2Te3 reported in −1 m−1 and σ = ~608 S cm−1 at 400 K) to its bulk form  of conventional TE materials have yielded great improvements, often showing an increase in −1) with many around 102 S cm−1. When comparing PEDOT:PSS (5% DMSO)/Bi2Te3 composite (945 S cm−1 at 298 K) to the conventional Bi2Te3 /Te nanorods) compared to the PTh class /Bi2Te3). The few composites reported with high polymer loadings (>60% polymer) show a lower 2Te3 composite (11.5 S cm−1) to pristine PANI , there is a clear disparity, with the composite possessing a much lowerWhen comparing several composites  to theirT/Bi2Te3 , an incrmaterial . ConversGraphene ,179.S = 210–1000 µV K−1), and are indeed more comparable to most polymers (1 µV K−1). All of the PTh-based composites have high S values. Of the N-based composites, those containing PANI have a more balanced S. Again, the relationship between S and S andS for the conventional/polymer composites is 20–190 µV K−1, with all composites having a p-type behavior. This differs from many of the conventional TEs used within the conventional/polymer composites, which are usually n-type when in their bulk form /Bi2Se3 (~188 µV K−1) followed by PEDOT/CNT/Bi2Te3 (~122 µV K−1). The latter has an S lower in magnitude than that reported for its conventional constituent Bi2Te3 (−1). Conversely, the addition of conventional TEs to polymers can considerably increase the magnitude of S, for example, PANI (70 wt. %)/Bi2Se3 composite has indeed a higher S than PANI (39.5 µV K−1), When considering the majority of conventional/polymer composites across both polymer classes , it is aulk form . This isulk form  compositi2Te3 (1400 µW m−1 K−2), which is far greater than any of those reported for the state-of-the-art polymers (max = 1270 µW m−1 K−2 for PP-PEDOT:Tos), and a number of the state-of-the-art inorganic materials (−1 K−2), the highest performing composites (where PF = ≥102µW m−1 K−2) are observed over almost all of the polymer loadings (5–80 wt. %) and seem to be highly dependent upon the conventional materials present. When comparing between polymer classes, PTh class report much higher PF  than the N class /Bi2Se3), although the PTh front-runner composite does also contain carbon. Overall, the PF values of the conventional/polymer composites are more comparable to the state-of-the-art polymers  to its inorganic constituent Bi2Te3  to PEDOT:PSS , compared to the PTh class (0.2–0.49). The highest ZT belongs to PEDOT/CNT/Bi2Te3 (0.49 at 325 K), which is a rather impressive increase compared to typical Bi2Te3 /Bi2Te3 expresses a low ZT of 4.3 × 10−3 at 300 K, far less than that of Bi2Te3  [The addition of polymers such as PANI, PEDOT:PSS, etc., to oxide materials is expected to yield similar improvements to the more explored science of creating composites via the addition of carbon allotropes to inorganic materials. In ons (RT) . Althougσ, S, From the available literature, it is clear that the creation of oxide/polymer composites is relatively underrepresented within TE research, with only a handful of review articles briefly mentioning them ,180,181.One could infer the optimal temperature operating range of the oxide/polymer composites using S. If we compare the 3, which shows an increase in −1 when compared to PANI alone /Al0.4ZnO composites. PANI/BaTiO3 (270 S cm−1) has a greater 3,−1, whereas PANI (84 wt. %)/Al0.4ZnO (25 S cm−1) shows a smaller 0.998Al0.02O, ~1370 S cm−1. Electrical conductivity,polymers , on avertituents . As dire0.5Ni0.5Fe2O4 shows an almost linear increase in 2O4 consisting of an initial increase in 3), and the NAr class /C (5 wt. %)/Ca3Co4O9).S and The addition of graphene and/or other highly conductive fillers to the oxide/polymer composites could be a promising strategy to improve 0.5Ni0.5Fe2O4 and PANI (80 wt. %)/SrTiO3). On the other hand, the p-type oxide/polymer composite, PANI (84 wt. %)/Al0.4ZnO (53.6 µV K−1), contains an n-type oxide, Zn0.998Al0.02O /Al0.4ZnO, as when compared to PANI, ~40 µV K−1 at 420 K /Liat 420 K , there iat 300 K , thus su0.5Ni0.5Fe2O4, shows the highest thermopower (S), comparable to the carbon-based composite Graphene (0.6 wt. %)/La0.067Sr0.9TiO3 , there is an increase in the magnitude of S as temperature increases. To summarise, the thermopower of oxide/polymer composites is comparable to those reported for polymers (S (331 µV K−1 at 383 K) while PANI alone is 39.5 µV K−1 at 420 K. When compared to oxide materials  to PANI (84 wt. %)/Al0.4ZnO (53.6 µV K−1). The n-type composite material containing the NAr polymer PPP, 0.95 wt. % PPP/Lir0.9TiO3 , with a polymers  and remaaterials , the mag2O4, which levels out and starts to decrease above ~290 K  has a much smaller PF compared to PEDOT:PSS  compared to PPy (0.32 µW m−1 K−2 at 380 K). In comparison to oxide materials (3Co4O9 (~62 µW m−1 K−2) in −1 K), as their PF values are in the range of those of conventional/polymer composites. The carbon filler-containing perovskite oxide composite, Graphene (0.6 wt. %)/La0.067Sr0.933TiO3  compared to PEDOT/TiO2-ZnO (ZT = 7.5 × 10−3 at 383 K).For oxide/polymer composites which report tituents , e.g., P3Co4O9 and Matrimid (10 wt. %)/C (5 wt. %)/Ca3Co4O9 allow us to compare between a composite with and without a carbon filler [S at 523 K, where S = ~140 µV K−1 and ~178 µV K−1 for the composite containing no filler and filler respectively, a reasonable improvement of 38.6 µV K−1 is observed with the addition of carbon-black (5 wt. %). This is perhaps attributed to the greater conductivity of this constituent. This is supported by −1 is shown on the addition of carbon-black (5 wt. %) yielding an −1, a great improvement compared to the oxide/polymer composite without filler −1. The −1 m−1 at 523 K for the composite with filler, while −1 m−1 at 523 K for that without. This results in a PF = 3.17 μW m−1 K−2 at 523 K for the filler containing material, a dramatic improvement upon the no filler composite, where PF = ~0.6 μW m−1 K−2. Hence providing evidence that the addition of filler to polymer composite materials can indeed improve TE properties. The composites Matrimid (10 wt. %)/Can filler . In FiguS and S. Therefore, the addition of both highly conductive filler and polymer to a high loading of oxide may be the recommended route to gain the most benefit from the creation of a composite. This should ensure a balance between all parameters and provide the most benefits from the disruption of phonon modes introduced by synergistic effects of the material’s interfaces.In general, strategies for improving oxide TEs aim to lower Oxide materials usually exhibit their optimal performance at high temperatures, whereas most conventional materials usually cannot operate within this range (>~700 K) . If the When pairing materials for the generation of composites, the temperature range of each constituent may need careful consideration. The vast majority of polymers operate more effectively in the mid-to-lower temperature ranges reported by oxides (<600 K); however, there are some exceptions such as PANI (base), PEDOT and PPy that are stable over a larger portion of the oxide temperature range (<800 K). Therefore, the exclusive use of doped polymers for applications at ~RT − 320 K may be suitable, while the polymer PPP may be suited for higher temperature applications (>700 K) or in circumstances where there is a high variation in temperature range. The additions of carbon-based fillers to a polymer/oxide composite may also impact the temperature range of stability. It could also bring the optimal operating temperature range down to lower temperatures opening up the operating temperature window of the individual oxide ,95. Mani


Due to the heterogeneity of breast cancer, current available treatment options are moderately effective at best. Hence, it is highly recommended to comprehend different subtypes, understand pathogenic mechanisms involved, and develop treatment modalities. The repurposing of an old FDA approved anti-malarial drug, amodiaquine (AQ) presents an outstanding opportunity to explore its efficacy in treating majority of breast cancer subtypes. Cytotoxicity, scratch assay, vasculogenic mimicry study, and clonogenic assay were employed to determine AQ’s ability to inhibit cell viability, cell migration, vascular formation, and colony growth. 3D Spheroid cell culture studies were performed to identify tumor growth inhibition potential of AQ in MCF-7 and MDAMB-231 cell lines. Apoptosis assays, cell cycle analysis, RT-qPCR assays, and Western blot studies were performed to determine AQ’s ability to induce apoptosis, cell cycle changes, gene expression changes, and induction of autophagy marker proteins. The results from in-vitro studies confirmed the potential of AQ as an anti-cancer drug. In different breast cancer cell lines tested, AQ significantly induces cytotoxicity, inhibit colony formation, inhibit cell migration, reduces 3D spheroid volume, induces apoptosis, blocks cell cycle progression, inhibit expression of cancer related genes, and induces LC3BII protein to inhibit autophagy. Our results demonstrate that amodiaquine is a promising drug to repurpose for breast cancer treatment, which needs numerous efforts from further studies.  Breast cancer is one of the most common type of cancers among women, making up to 25% of all new cancer diagnoses with continuously increasing incidence rates ,2. AccorWith recent advancements in the cancer therapeutics, multiple treatment choices are available for breast cancer treatment based on the type and aggressiveness of the cancer that have resulted in overall increase in patient survival  ,11. WhilDeveloping a new drug is a lengthy and cumbersome process, and requires a lot of investment ,17, not Mechanism based repurposing of an old FDA approved drug presents an outstanding opportunity to explore its efficacy in treating breast cancer . For insIn the project, we hypothesize that AQ exerts its anticancer efficacy in majority of breast cancer subtypes namely ER, PR positive breast cancer types; triple negative breast cancer (TNBC); and human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell types due to its ability to induce apoptosis and to inhibit autophagy in various cancers. In this project, we aim to identify the breast cancer types responsive to AQ treatment and to explore the efficacy of amodiaquine against breast cancer through multitude of in-vitro and ex-vivo cell models.We have recently demonstrated the anti-cancer effects of amodiaquine in non-small cell lung cancer cells . We test50 of 11.5 ± 6.5 µM (MCF-7), 8.2 ± 2.8 µM (MDAMB-231), 24.0 ± 2.2 µM (BT-549) and 16.0 ± 3.9 µM (SK-BR-3). The results have demonstrated the varied cytotoxicity potential of AQ against different breast cancer types where dose-dependent inhibition of cell proliferation was recorded. Lower IC50 values have been observed for AQ in case of MCF-7 and MDAMB-231 compared to BT-549 and SK-BR-3. The observed differences in cytotoxicity may be due to the distinct properties of different subtypes of breast cancer. AQ may have the capability to exert its anti-cancer efficacy via Estrogen and progesterone receptors predominantly as both MCF-7 and MDAMB-231 cell lines are HER2 negative. Hence, it is assumed that AQ possesses superior cytotoxic potential against certain breast cancer types.As observed in cytotoxicity studies , amodiaqp < 0.05, Control vs. AQ 25 μM—p < 0.01). From Scratch assay is a well-established method to access cell-cell interaction, and cellular migration . For theVasculogenic mimicry assay was performed to evaluate the inhibitory effect of AQ on vascular network formation of aggressive MDAMB-231 breast cancer cell line. Formation of 3D channel-like networks which are representative of the initial stages of vasculogenic mimicry were photographed under an inverted microscope (10×)  after 8 h. Cells were observed by phase-control microscopy during after the incubation period. As shown in : p < 0.01, Control vs. AQ 15 µM: p < 0.0001, Control vs. AQ 25 µM: p < 0.0001, AQ 7.5 µM vs. AQ 15 µM: p < 0.01, AQ 7.5 µM vs. AQ 25 µM: p < 0.001). In case of MDAMB-231, % colony growth was found to be 26.0 ± 3.0%,4.5 ± 2.7% and 0.6 ± 0.3% for AQ 7.5, 15 and 25 µM, respectively, considering number of colonies to be 100% in drug free treatment control wells. . These data clearly representative of AQ’s efficacy in eliminating the possibility of tumor relapse, from single cancer cells left behind following chemotherapy and surgical intervention  and 0.8 ± 0.4 mm3 (multiple dose) on an average on day 0 before dosing with respective treatments. It was found that spheroid volumes on day 10 after single dosing were 10.2 ± 2.0 mm3 (Control), 6.7 ± 1.5 mm3 (AQ 7.5 µM), 1.0 ± 0.5 mm3 (AQ 15 µM), 0.1 ± 0.3 mm3 (AQ 25 µM), showing dose-dependent reduction in tumor mass and viability (3 (Control), 12.6 ± 1.3 mm3 (AQ 7.5 µM), 5.2 ± 2.2 mm3 (AQ 15 µM) and 0.3 ± 0.2 mm3 (AQ 25 µM)  B. 3 (single dose) and 0.8 ± 0.4 mm3 (multiple dose) on an average on day 0 before dosing with respective treatments. It was found that spheroid volumes on day 10 after single dosing were 2.0 ± 0.2 mm3 (Control), 2.2 ± 0.1 mm3 (AQ 7.5 µM) and AQ 15 and 25 µM treated spheroids were completely dissipated leaving no distinct spheroid mass which represent spheroid volumes of <0.05 mm3 . In case of multiple dosing, spheroid volumes on day 10 were found to be 1.9 ± 0.2 mm3 (Control), 1.8 ± 0.1 mm3 (AQ 7.5 µM), 0.2 ± 0.1 mm3 (AQ 15 µM) and <0.05 mm3 with AQ 25 µM treatment, as seen in : p < 0.0001, Control vs. AQ 25 µM: p < 0.0001). At the same time, MDAMB-231 spheroid volumes were found to be 0.7 ± 0.3 mm0.05 mm3 C. StatisFrom the observed efficacy in both spheroid models, it was confirmed that treatment with AQ was able to reverse the tumor formation in an in-vitro setting, following a customizable dose-dependent manner. Measuring the extremities of the tumor/spheroid may not reveal the events at the core of the tumor, traditionally considered the hard-to-reach regions for cancer therapeutics. CellTiter-Glo luminescent cell viability assay was performed to compare counts of viable cells in treated 3D single- and multiple-dose spheroids on day 10 after microscopic quantification. As seen with volume measurement, AQ treated MCF-7 spheroids exhibited significantly reduced proportion of viable cells compared to control in a dose-dependent manner. % cell viability results are represented in As the physiological and 3D tumors represent biological matrices, it is imperative for the drug to penetrate through tumor’s microenvironment, in order to demonstrate its full therapeutic potential. Therefore, it is necessary to quantify dead cell and live cell portions out of a spheroid mass. Representative Live-dead cell assay images are represented in n = 3–5 trials). Here, the right shift of the scatter plot can be clearly observed in the case of AQ (7.5 µM and 15 µM) compared to that of the control (p < 0.01). There was also a significant difference between treatments with two different concentrations of AQ (p < 0.01: AQ 7.5 µM vs. 15 µM). The results indicate the ability of AQ to induce early-stage apoptosis even at both concentrations of 7.5 µM and 15 µM which led to the highest proportion of total % apoptotic cells.The Muse Cell Analyzer determined the extent of apoptosis in MDAMB-231 cells incubated with AQ at 7.5 and 15 µM concentration for 24 h. The apoptotic profile of MDAMB-231 cells after incubating with respective treatments is shown as scatter plots in  control A. As quap < 0.05) and AQ 7.5 µM vs. AQ 25 µM: (p < 0.05)) with an increase in AQ dose was shown in To study the mechanism of improved efficacy of amodiaquine, cell cycle analysis was conducted by measuring DNA content by propidium iodide (PI) staining method using flow cytometry. PI staining is very rapid, reproducible, and reliable method which can be used for estimation of cell cycle parameters. As can be seen in quantification presented in p < 0.05) and MDAMB-231 cell lines  after AQ treatment as compared to their respective control blots  (β-catenin and LC3BII  play a m0 ± 0.5) E,F.To further elucidate the potential mechanism for AQ’s efficacy in breast cancer therapeutics, we performed gene expression analysis in MDAMB-231 cell line after treatments with 0–20 µM concentrations of AQ. The results  show thaBreast cancer being the most common type of cancer among women, extensive investigations have been carried out to explore novel treatment possibilities . Even thIn the current era of escalating need for new drugs against cancer, there is an emerging interest in identifying new uses for old drugs especially anti-malarial drugs ,46. For We selected a panel of breast cancer cell lines representing major clinically relevant subtypes, responsible for resistance of this disease to chemotherapy. We profiled the activity of AQ, in terms of cell viability inhibition, cell migration and colony growth inhibition. Cell viability is an important toxicity assay parameter and is directly associated with the toxic effects of a drug . AQ induCell migration and invasion are important processes in tumor development and metastasis . Howeverin-vivo tumor conditions, it could be further tested in preclinical studies. Two-dimensional in-vitro assays performed may or may not necessarily mimic in-vivo conditions due to the complexity of in-vivo tumor models and their uncontrolled cell growth, solid tumoral mass hindering the penetration of drugs and varied tumor microenvironment . Thus, eFurthermore, the AQ was further explored to illustrate its mechanism of action through estimation of cell cycle parameters using flow cytometry. It has been observed that amodiaquine is capable of causing cell arrest in S-phase, hence hindering them from entering mitotic phase as can be seen in To our knowledge, this is the first study reporting the efficacy and detailed molecular mechanism of action of amodiaquine for treatment of breast cancer. Taken together, supporting evidence from various in-vitro and ex-vivo studies demonstrated the superior anti-tumor activity of amodiaquine for the treatment of breast cancer.2/37 °C. BT-549 was maintained in RPMI-1640 medium supplemented with 10% FBS , 1% sodium pyruvate , 1% penicillin-streptomycin , 0.023 U/mL of Gibco™ Insulin, human recombinant zinc solution ). SK-BR-3 was maintained in Hyclone McCoy’s 5A Medium ) supplemented with 10% FBS  and 1% penicillin-streptomycin . 3--2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), crystal violet dye, and 16% paraformaldehyde (PFA) solution were purchased from Fisher Scientific . All molecular biology kits and supplies were purchased from other commercial vendors which are listed at appropriate places throughout the manuscript. MCF-7 (HER2 positive), MDAMB-231 (TNBC), BT-549 (TNBC), and SK-BR-3 (HER-2 positive) breast cancer cell lines were obtained from ATCC . MCF-7, and MDAMB-231 were maintained in DMEM medium  supplemented with 10% FBS  and penicillin-streptomycin  at 5% COAQ was evaluated for its cytotoxicity efficacy in four different breast cancer cell lines: MCF-7, MDAMB-231, SK-BR3, and BT-549 as reported earlier with slight modifications ,32. DetaIn-vitro scratch assay was used to study the cell migration. Briefly, scratches were created on a confluent cell monolayer. The cells on the edge of the scratch will migrate toward the center to close the scratch, thus establishing new cell-cell contacts. The assay was performed on MDAMB-231 cell line as previously reported ,61. Deta® RGF BME  was added to each well of a 96-well plate and incubated at 37 °C for 60 min to allow the BME to gel. Briefly, MDAMB-231 cells were seeded at 2.0 × 104 cells/well on this BME-coated 96-well plate after preparing dilutions with varying concentrations  of AQ. Cells were incubated for 8 h at 37 °C/5% CO2. Phase contrast images were taken using an inverted microscope  with 10× magnification, and effect of AQ on tube formation was qualitatively observed from the images.Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Recent studies have found that some highly aggressive tumor cells generate vessel-like channels in the absence of endothelial cells or fibroblasts ,63. ThesUsing this assay, the effectiveness of the AQ towards colony inhibition was determined in MCF-7 or MDAMB-231 cells. Protocols reported previously  was brieCompared with two-dimensional (2D) monolayer culture, breast cancer spheroids more accurately reflect the complex microenvironment in-vivo . Many of®, Promega, Madison, WI, USA) on MCF-7 and MDAMB-231 spheroids on 10th day according to manufacturer’s protocol. After imaging (10× magnification), 100 µL of medium was removed and replaced with 100 µL of CellTiter-Glo® reagent in each well. The contents were mixed for 2 min, followed by 30 min incubation at room temperature. The luminescence was measured using Spark 10 M plate reader . The results were presented as % cell viability  and compared with fresh media blank controls.CellTiter-Glo cell viability assay was performed using a commercially available kit . According to manufacturer’s protocol, live-dead cell study was performed on treated MCF-7 and MDAMB-231 spheroids on day 10th of both single and multiple dosing in the therapeutic model. Briefly, after complete removal of media from the wells; 100 μL of staining solution ) was added to treated spheroids; in order to give the green/red fluorescent staining for viable and dead cells, respectively. Plate was then incubated at room temperature for 45 min in dark. Images were captured at 4× magnification using fluorescence microscope . 6 cells/mL; and 150 µL of Annexin V/dead reagent and 100 µL of a single cell suspension were mixed in a microtube thoroughly by vortexing for 5 s, followed by incubating in the dark for 20 min at room temperature. Cells were then analyzed using the Muse cell analyzer . The apoptotic ratio was determined by identification of four populations: (i) non-apoptotic cells, not undergoing detectable apoptosis, Annexin V (−) and 7-AAD (−); (ii) early apoptotic cells, Annexin V (+) and 7-AAD (−); (iii) late apoptotic cells, Annexin V (+) and 7-AAD (+); and (iv) cells that have died through non-apoptotic pathway, Annexin V (−) and 7-AAD (+).Apoptotic cell distribution of MDAMB-231 cells was assayed by using the MUSE Annexin V & Dead Cell Kit  according to the manufacturer’s instructions. Briefly, MDAMB-231 cells were seeded  in a 24 well plate and incubated overnight. The next day, the media was replaced with AQ , or fresh media. After 24 h, treatments were removed, and cells were collected through trypsinization followed by washing with PBS twice. Cell suspension was diluted with growth media to a concentration of 0.5 × 10® software.Following treatment for 72 h time period, cell cycle was studied using propidium iodide (PI) assay with flow cytometry using reported protocols with slight modifications [6 cells per petri dish and were treated with AQ  at 37 °C/5% CO2. After treatment, the cells were collected and lysed with 1% Triton® X-100  and 1% Halt™ Protease and Phosphatase Inhibitor Cocktail  in PBS and bath sonicated for 1 h at 4 °C. Samples were centrifuged for 15 min at 4°C at 15,000 rpm and lysates were collected. Cell lysate protein was quantified by DC™ Protein Assay Kit . The samples were mixed with 2× Laemmli buffer Bio-Rad, Hercules, CA, USA) and 2-mercaptoethanol, and denatured at 110 °C for 10 min. For Western blot analysis, 10 µg protein was loaded and separated on 4–20% Mini- PROTEAN® TGX™ Precast Protein Gels  and transferred to Trans-Blot® Turbo™ Midi PVDF membranes  using a Bio-Rad PowerPac™ Basic Power Supply and Trans-Blot® TurboTM system. The membranes were blocked with 5% bovine serum albumin in PBS and probed with corresponding primary antibodies (1:1000 dilution) and kept for overnight shaking at 4 °C. The following antibodies were used: β-actin ; LC3B-II  and β-catenin . Membranes were then incubated with corresponding secondary HRP-conjugated antibodies: goat anti-mouse (#31430) poly-HRP  and goat anti-rabbit (#32260)   for 1 h at room temperature and subjected to Western Bright chemiluminescence . Protein signals were detected on the membranes and were quantified using the chemiluminescent imaging by the Omega Lum™ G Imaging System .MCF-7 and MDAMB-231 cells were plated 1 × 10Triple-negative breast cancer cell line MDAMB-231 was used for real-time quantitative polymerase chain reaction (RT-qPCR) experiments. 1 million cells/well were seeded in a 6-well plate as described above followed by culturing for 24 h. Confluent (80%) cells were treated with different concentrations of amodiaquine (0–20 µM) for 12 h in duplicates. Following the drug treatment, cells were trypsinized, washed twice with ice-cold PBS, and cell pellets were collected. Total RNA was extracted from cells using the Qiagen RNeasy plus mini kit  according to the manufacturer’s instructions. Briefly, cell pellets were homogenized using RLT buffer, and cell lysates were passed through a gDNA elimination column to remove any genomic DNA from the lysate. RNA was precipitated using 70% ethanol treatment of the lysate followed by binding on the RNeasy mini column. Column bound RNA was washed once with buffer RW1 followed by washing twice with buffer RPE before eluting with water. The eluted RNA was quantified using a Nanodrop spectrophotometer , and purity of RNA was determined by the absorbance ratio of 260/280 nm. Equal amount of total RNA was used to synthesize single stranded cDNA using High-Capacity cDNA Reverse Transcription Kit . Briefly, RT buffer (10×), 100 nM dNTP mix, RT random primers (10×), RNase inhibitor, and MultiScribe Reverse Transcriptase were used in a PCR reaction to convert RNA to cDNA. PCR reactions were carried out using the MiniAmp Thermal cycler  at standard cDNA amplification cycle. t-test for expression fold difference of individual genes. RT-qPCR reactions were performed using the SYBR Green dye technology  to determine the gene expression changes in response to drug treatments. Specific gene specific primers were designed  and usedn = 3 unless otherwise mentioned. Three trials of cytotoxicity studies were performed for each control or treatment with n = 6 for each trial. All data were evaluated by unpaired student’s t-test or one-way ANOVA followed by Tukey’s multiple comparisons test, using GraphPad Prism software .All data were addressed as mean ± SD or SEM, with Research targeting finding new anti-cancer therapies is prompted by cancers’ high mortality rate. Anti-malarial drugs have taken their place in the research arena as new, effective medicines in treating various breast cancer sub types. The current study establishes the overall utility of an antimalarial drug, AQ, in treatment of breast cancer by evaluating its anticancer activities through several in-vitro cell culture studies as well as 3D spheroid models. Altogether, AQ has demonstrated superior anti-tumor properties illustrated by its potential to inhibit autophagy, induce apoptosis, and cause cell cycle arrest. Even though animal studies were not employed to assess AQ’s efficacy, recent studies have compared 3D spheroids and in-vivo studies, and have reported similar outcomes in both cases ,74. Furt


Vitamin A is one of the important micronutrients involved in various biological functions. Numerous studies have suggested a link between vitamin A (retinoic acid) and type 2 diabetes (T2D). However, the functional role of vitamin A receptors  in β-pancreatic cells is not clear yet. In this study, we performed bioinformatics and functional experiments in human islets and INS-1 cells in order to evaluate the potential role of RARβ on insulin secretion and pancreatic β-cell function.trans-retinol, and type 2 diabetes (T2D). However, the functional role/expression of vitamin A receptors  in pancreatic β-cells is not clear yet. Accordingly, we performed a series of bioinformatics, molecular and functional experiments in human islet and INS-1 cells to evaluate the role of Rarβ on insulin secretion and pancreatic β-cell function. Microarray and RNA-sequencing (RAN-seq) expression analysis showed that RARα, β, and γ are expressed in human pancreatic islets. RNA-seq expression of RARβ in diabetic/hyperglycemic human islets (HbA1c ≥ 6.3%) revealed a significant reduction (p = 0.004) compared to nondiabetic/normoglycemic cells (HbA1c < 6%). The expression of RARβ with INS and PDX1 showed inverse association, while positive correlations were observed with INSR and HbA1c levels. Exploration of the T2D knowledge portal (T2DKP) revealed that several genetic variants in RARβ are associated with BMI. The most associated variant is rs6804842 (p = 1.2 × 10−25). Silencing of Rarβ in INS-1 cells impaired insulin secretion without affecting cell viability or apoptosis. Interestingly, reactive oxygen species (ROS) production levels were elevated and glucose uptake was reduced in Rarβ-silenced cells. mRNA expression of Ins1, Pdx1, NeuroD1, Mafa, Snap25, Vamp2, and Gck were significantly (p < 0.05) downregulated in Rarβ-silenced cells. For protein levels, Pro/Insulin, PDX1, GLUT2, GCK, pAKT/AKT, and INSR expression were downregulated considerably (p < 0.05). The expression of NEUROD and VAMP2 were not affected. In conclusion, our results indicate that Rarβ is an important molecule for β-cell function. Hence, our data further support the potential role of VA receptors in the development of T2D.Various studies have suggested a link between vitamin A (VA), all- As our understanding of the pathogenesis of T2D has been dramatically increased, the interaction between genetic susceptibility and lifestyle, including diet, are well-established as the main factors in the occurrence of the disease [Type 2 diabetes mellitus (T2D) is a multifactorial and heterogeneous metabolic disorder characterized by a higher glucose level due to insufficient insulin production, action, or both. The disease’s prevalence is increasing globally and is expected to affect more than 550 million people in the next 10 years  along with three retinoid-X receptors  ,20. TranTogether, these data indicate that RAR signaling is essential to maintaining β-cell function and mass. However, the role of Rarβ and γ in insulin secretion is not clear. Additionally, the expression of RARs in murine pancreatic cells are well documented; however, expression in human pancreatic islets is not mature ,24. Thus® HG Waltham, MA, USA) were implemented according to the Affymetrix standard protocol [Human pancreatic islet RNA-seq expression data were retrieved from a publicly available database . RNA-seq data set includes pancreatic islets from 77 cadaver donors, which includes 50 nondiabetic (HbA1c < 6%) and 25 diabetic (HbA1c ≥ 6%) donors [protocol . Pancreahttps://t2d.hugeamp.org (accessed on 16 May 2022)), which consists of 310 datasets and 339 traits, was used to analyze the association of genetic variants within RABβ and genetic information that is linked with T2D and associated traits.The type 2 diabetes (T2D) knowledge data portal  were cultured in RPMI 1640 as described previously . The cel2 for 72 h. The concentration of siRNA is 40 nM in 24-well cell culture plates . Transfected cells were washed twice with 1 mL secretion assay buffer (SAB) at pH 7.2 having 2.8 mM glucose and then incubated for 2 h in a 2 mL new SAB (with 2.8 mM glucose). Next, an additional incubation step for 1 h was performed in 1 mL SAB with 2.8/16.7 mM glucose.Pancreatic β-cells (INS-1) were silenced as in a previously described method ,28 usingAfter 48 h of transfection, GSIS (glucose-stimulated insulin secretion) was investigated. Transfected INS1 cells were incubated in SAB buffer containing 0.2% BSA at pH 7.2 with 2.8 mM glucose for 2 h. GSIS was performed by incubating cells in 1 mL SAB containing 2.8 mM glucose along with the secretagogue mentioned previously ,28. InsuWestern blot analysis was performed as in a previously described protocol ,28 with 5 INS-1 cells were seeded in a 96-well plate for 24 h and transfected as previously described in the Briefly, 2 × 10−ΔΔCt method.Total RNA was extracted by RNA Kit  and quantity/quality was measured on Nanodrop . RevertAid H Minus First Strand cDNA Synthesis Kit was used to produce cDNA . Gene expression quantification was performed by TaqMan assay  in qPCR Quantstudio3 machine from Applied Biosystems  with specific primer probes for Ins2 (Rn01774648_g1), Ins1 (Rn02121433_g1), Pdx1 (Rn00755591_ m1), Mafa (Rn00845206_g1) Insr (Rn00690703_m1), Glut2 (Rn00563565_m1), Gck (Rn00561265_m1), and Hprt1 (Rn01527840_m1). Expression of Snap25, Vamp2, NeuroD, Rarβ, Rarγ, and Rarα were perfumed by using the following SYBR-Green primers: Snap25 , Vamp2 , NeuroD , RARβ , RARα , RARγ . Hprt  was used to normalize the gene expression. The relative gene expression was measured by 2Transfected INS-1 cells were cultured in a complete RPMI for 24 hours as previously described ,28. NextTM III) and analyzed by FlowJo_v10.8.1 software .Glucose analog 2-NBDG, from Invitrogen (Cat. #N13195), was used to assess glucose uptake according to the manufacturer’s procedure. Briefly, siRarβ cells were incubated with 100 µM/mL of 2-NBDG (dissolved in ethanol) and incubated for 1 h in a humidified incubator at 37 °C. Next, the harvested cells were washed two times with ice-cold PBS, then centrifuged at 1800 rpm for 5 min at 4 °C. Finally, cells were re-suspended in 250 µL of ice-cold PBS. Stained cells were immediately quantified by flow cytometry  was used. The manufacturer’s guidelines were followed when performing the H2O2 assay. Briefly, 15,000 INS1 cells were seeded in a 96-well plate overnight; after 24 h transfection, 20 µL of H2O2 substrate was added to every well and incubated at 37 °C in a humidified incubator in a fixed atmosphere of 5% CO2 for 4 h. After incubation, 0.1 mL of ROS-Glo detection reagent was added to the experimental wells and was kept for 20 min at room temperature. Immediately after, the fluorescent intensity was measured by using a luminometer plate reader.To measure the Ht-tests or nonparametric Mann–Whitney tests were used for differential expression analysis between diabetic versus non-diabetic islets. The correlation analyses were performed by nonparametric Spearman’s correlation test. Statistical analyses were performed on GraphPad Prism, V8 Software, San Diego, CA, USA). In all analyses, differences were significant at * p < 0.05, ** p < 0.01 or *** p < 0.001.Student n = 1) (Microarray and RNA-seq expression data from human pancreatic islets were used to describe the expression profile of RARs. As shown in n = 1) C.p < 0.05) in diabetic islets (HbA1c ≥ 6.0%) compared to nondiabetic islets (HbA1c < 6%)  D. Expres1c < 6%) E,F. Addi1c < 6%) G,H, wher1c < 6%) I,J. No cp  <  1 × 10−8 for the association with BMI, with rs6804842 being the strongest associated genetic variant. A list of the top 10 associated genetic variants in RARβ with BMI are shown in Further, we evaluated whether RARβ contains any genetic variants associated with T2D or related traits in the T2D knowledge portal. Interestingly, we found a strong association between several genetic variants in RARβ and BMI in the GIANT UK Biobank GWAS. Accordingly, 103 genetic variants were shown to pass the significance threshold at p < 0.05) relative to the negative control (p < 0.05) and at stimulation level  after 1 h stimulation compared with control cells (p < 0.05) (p < 0.05) (p > 0.05) on the proportion of apoptotic cells  out of the total number of Rarβ-silenced cells compared to 5% with the control cells. These results were further confirmed by the cell viability test, which showed no significant (p > 0.05) effect of siRarβ on cell viability compared to the negative control cells (p < 0.05), as evidenced by the increased positive rate of fluorescence and mean fluorescence intensity in siRarβ cells compared to control cells. On the other side, a substantial reduction in glucose uptake  was observed in siRarβ cells compared to control cells  E. Insuli < 0.05) F. Then, ol cells B. To thiol cells D.Rarβ silencing on genes involved in pancreatic β-cell function, we analyzed the mRNA expression of insulin biosynthesis genes. As illustrated in Ins1 was significantly (p < 0.05) reduced  (p < 0.05) (p < 0.05) in Rarβ-silenced cells compared to negative control cells  were significantly reduced in siRarβ cells compared to the negative control (p < 0.05) (Insr) was also downregulated non-significantly (p > 0.05) (p > 0.05) (p > 0.05) (p < 0.05) (p < 0.05) (p < 0.05)  C,E. Geneol cells F,G. More < 0.05) H,I. Howeficantly N. Protei > 0.05) A, PDX1(~ > 0.05) B, and p- > 0.05) D. Moreov < 0.05) E, GCK (~ < 0.05) F, and IN < 0.05) H was redS-1 cell C,G.Several studies have demonstrated that retinoids and VA possess anti-obesity and anti-lipogenic activities via transcriptional regulation of multiple relevant genes, mainly in the liver and adipose tissue ,31,32 HoSeveral points are worth mentioning; first, the expression of RARs is well-documented in animal models or cell lines ,21,23, bSecondly, the reduced expression levels of RARβ in diabetic islets is a novel finding D. WhetheThirdly, the co-expression correlation of RARβ with the Insulin gene or HbA1c levels was surprising. Considering the expression of RARβ is downregulated in diabetic islets, we expected the expression RARβ would positively correlate with Insulin gene expression and negatively with HbA1c, but that was not the case. Possible explanations for this finding are a change in mRNA expression of the tested genes due to long-term hyperglycemia status or epigenetic regulations that control RARβ gene expression . AdditioAs determined, silencing of the Rarβ gene in INS-1 cells impaired insulin release without affecting cytotoxicity or apoptosis E. ExpresTo gain more insight into the molecular mechanisms of how Rarβ diminished insulin secretion, we investigated the expression of several β-pancreatic cell genes mainly involved in insulin biosynthesis, glucose sensing, insulin action, and insulin release. Previously, the retinoic acid-response element was anticipated to stimulate insulin mRNA levels in human islets . In lineFurthermore, expression assessment at the mRNA or protein levels of the β-cell transcription factors showed significantly decreased Pdx1, Mafa, and Neurod1 . HoweverGlut2 and Gck are two major players in glucose-sensing machinery that respond to physiological blood glucose changes in β-pancreatic cells. Several studies reported that any defect in glucose-sensing machinery would impair insulin secretion, which causes severe hyperglycemia ,43. In tInterestingly, the reduced expression of GLUT2 was accompanied by decreased glucose uptake efficacy, which is an essential driving factor for insulin release . Thus, oIn mammals, there are three isoforms of Akt  which play divergent functions. However, it is well known that Akt2 is a crucial element in insulin signaling . Akt actAlthough the strength of this study is the combination of genetics data through bioinformatics analysis with functional data, it is worth noting that this study has certain limitations. We used only an in vitro model, which cannot mimic human physiology. Second, the RNA-sequencing data addressed the expression of RARs in human pancreatic islets instead of sorted β-cells or other endocrine cells. Third, we focused only on Rarβ; other RARs might similarly affect pancreatic β-cell function. It will be of great interest to perform further investigations to dissect the role of Rarβ in other tissues, such as muscles and liver, and the association with glucose metabolism.In conclusion, the current data suggest that VA  and its receptors could be important for glucose metabolism and pancreatic β-cell dysfunction. In addition, our data also raise the possibility of using Rarβ as a drug target for T2D.


Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins.A raw starch-degrading α-amylase from B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter PspoVG–PspoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (PgroE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL·h.AmyZ1 secretory production was achieved in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading α-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.This is the first report of recombinant expression of AmyZ1 in The online version contains supplementary material available at 10.1186/s12934-022-01855-9. HoweverPontibacillus sp. ZY (named AmyZ1) with broad substrate specificity towards different raw starches at low temperatures (about 35 °C) and higher specific activity than other reported RSDAs [Escherichia coli as a recombination production host, AmyZ1 primarily presents as insoluble inclusion bodies, thus hampers its industrial application. Therefore, choosing a suitable expression host for AmyZ1 soluble extracellular production is beneficial to promote its industrial application.Thus far, although many RSDAs have been identified, little of them exhibit high specific activity toward raw starches . In addied RSDAs . Such prE. coli, Bacillus subtilis is a Gram-positive model bacterium with the advantages of strong protein secretion ability, easy to achieve soluble extracellular expression of proteins, and non-pathogenicity [B. subtilis has been an attractive strain for various industrial enzyme production [B. subtilis, such as secretion of multiple proteases, lack of strong promoter and efficient signal peptide (SP), and inefficient translation [B. subtilis, such as host strain optimization [Compared to genicity . Therefooduction . Howevernslation –13. Manymization , promotemization , and tramization .B. subtilis mainly include constitutive promoters and inducible promoters. Compared with constitutive promoters, inducible promoters have less influence on the growth of strains because they can control the expression cycle of target proteins. In past decades, many kinds of inducible promoter systems have been explored, such as tightly inducible expression systems induced by chemicals [B. subtilis were divided into four classes  [B. subtilis. For example, the extracellular amidase activities mediated by phase-dependent promoters PlytR (class III), PspoVG (class II), PaprE (class I), PyvyD (class II), PftsH (class II), and PamyE (class III) were 1.22-, 1.41-, 1.39-, 1.38-, 1.29-, and 1.46-fold higher than that mediated by widely used constitutive promoter P43, respectively [As a transcription-related regulatory factor, the promoter plays an important role in protein production . Currenthemicals  and autohemicals . Comparehemicals . In prevy phase) . To dateB. subtilis [B. subtilis using the dual-promoter systems [Bsamy-PBaamy, the alkaline serine protease (BcaPRO) activity in B. subtilis was 2.1- and 1.5-fold higher than those of strains with the promoters PBsamy and PBaamy, respectively [HpaII-PgsiB was 2.3- and 2.2-fold higher than those of the promoters PHpaII and PgsiB, respectively [Current research shows that constructing tandem promoters based on single promoters is an effective method to obtain novel high-efficiency strong promoters suitable for improving the extracellular expression of target proteins in subtilis . The eff systems , 23. Forectively . Likewisectively .B. subtilis by mutating the promoter core regions [2069M, obtained by improving the − 35 and − 10 regions of the promoter P2069, was twofold higher than that mediated by the promoter P2069 [P3510, obtained by changing the − 35 and − 10 regions of the promoter Pylb, was ninefold higher than that mediated by the promoter Pylb [In addition to the existing promoters, mutation of the promoter core regions such as − 35, − 16, − 10, and + 1 regions are also a practical approach to obtain the novel efficient promoters . Current regions . For exaer P2069 . The β-gter Pylb .B. subtilis [B. subtilis can be improved by optimizing the translation initiation efficiency [B. subtilis [Apart from optimizing the transcription level of target genes, improving its translation efficiency is also an effective strategy to enhance the extracellular expression of target proteins in subtilis . Many stsubtilis . Previouficiency , 29. Forficiency . Nowadaysubtilis .B. subtilis. Then, AmyZ1 production was enhanced by promoter engineering strategies, including screening phase-dependent promoters, constructing dual-promoters, and modifying promoters. Next, the 5′-proximal coding sequence predicated by UTR Designer was used to optimize translation initiation efficiency of the AmyZ1 coding gene. Finally, the resulting B. subtilis recombinant strain was cultured in 3-L fermenters to verify its ability to produce AmyZ1. Sketch map of the above strategies could see in Fig. In this study, AmyZ1 was first expressed extracellularly in E. coli existed in the form of inclusion bodies [B. subtilis WB600 with the recombinant vector pBHE, which contained the gene AmyZ1. After 48 h of fermentation in shake flasks, the extracellular AmyZ1 activity of BZ1 was 462.9 U/mL. Then, the AmyZ1 was successfully purified using the Ni-NAT system from the culture supernatant, which was verified by SDS-PAGE  [E. coli [B. subtilis.The biochemical characterization of recombinant AmyZ1 produced by industry . To veri21 U/mg) . This di[E. coli , which mB. subtilis are 7.0 and 40 °C, respectively , the widely used constitutive promoter P43 (σAB), and four endogenous phase-dependent promoters including PspoVG , PsecA , PodhA , and PlytR  in B. subtilis were investigated, respectively, to obtain an efficient promoter. The six plasmids containing different promoters pBHE (PgroE), pBHP (P43), pBHS (PspoVG), pBHA (PsecA), pBHO (PodhA), and pBHL (PlytR) were constructed , which was 2.06- and 1.36-fold higher than those of strains BZ1 and BZ2 containing PgroE (462.9 U/mL) and P43 , respectively. Similar to our results, the amidase activity and pullulanase activity mediated by promoter PspoVG were 1.41- and 1.37-fold higher than those mediated by promoter P43, respectively [spoVG might be a strong promoter that could be used in other expression systems.As shown in Fig. ectively , 32, sugAmyZ1 mRNA levels were determined by quantitative real-time PCR. The AmyZ1 mRNA levels of the strain BZ3 (PspoVG) were 3.03-, 3.17-, and 1.06-fold higher than those of strain BZ1 at 12, 24, and 48 h (p < 0.05), respectively  were also 1.69-, 1.76-, and 1.74-fold higher than those of strain BZ2 containing widely used promoter P43 at 12, 24, and 48 h, respectively. These results were consistent with the corresponding AmyZ1 expression levels   decreased first then increased , pBHPS (P43-PspoVG), and pBHSS (PspoVG-PspoVG), respectively. These plasmids were then transformed into B. subtilis WB600, generating the strains BZd1, BZd2, and BZd3, respectively.To explore the application potential of using dual-promoter engineering to improve the expression level of AmyZ1 in promoter , the prospoVG-PspoVG showed the highest activity, which was 1.19-fold higher than that of strain BZ3  containing promoter PspoVG. SDS-PAGE analysis further confirmed the above results  or dual-promoters (BZd1 and BZd2) during the whole fermentation stage  activity mediated by the dual promoter PamyE-P43 (99.34 U/mL) was 1.8- and 2.63-fold higher than those mediated by promoters PamyE (55.19 U/mL) and P43 (37.78 U/mL), respectively, and the Bm-Ami mRNA transcription level of the strain containing dual-promoter PamyE-P43 was higher than those of strains containing promoters PamyE and P43 throughout the fermentation stage [As shown in Fig. lts Fig. B. Furtheon stage . TherefogroE-PspoVG), BZd2 (P43-PspoVG), and BZd3 (PspoVG-PspoVG) containing dual-promoters were not the simple addition of the results seen with strains BZ1 (PgroE), BZ2 (P43), and BZ3 (PspoVG) containing the corresponding single promoters. In addition, only dual-promoter PspoVG-PspoVG mediated higher AmyZ1 activity than that mediated by PspoVG. In previous work, it was reported that only two of six dual-promoters mediated higher aminopeptidase activities than that mediated by the best single promoter and it indicated that the promoter origin influenced the cooperativity of the dual-promoter [In this study, the extracellular AmyZ1 activities of strains BZd1 (Ppromoter . BesidesspoVG-PspoVG) decreased  were all decreased [Compared with the other strains containing single promoters, however, the biomass of strain BZd3  with poor performance at stationary phase [AmyZ1 mRNA level of the best dual-promoter PspoVG-PspoVG at 48 h was highest throughout the fermentation stage  with the promoter PspoVG. What is more, the AmyZ1 mRNA level of strain BZd1 was also lower than that of strain BZ3 at 36 and 48 h (p < 0.05) Similarly, the extracellular aminopeptidase (AP) activities of strains BSG21, BSG23, BSG25, and BSG26 containing the dual-promoters PHpaII-PHpaII, PHpaII-P43, PHpaII-Pluxs, and PHpaII-PaprE were 3.6, 5.1, 9.2, and 15.7 U/mL, respectively, lower than that of the strain BSG11 (31.9 U/mL) containing the single promoter PHpaII [B. subtilis under certain circumstances. It may be caused by factors such as the types of sigma factors and 5'-untranslation region sequences of genes [However, the extracellular AmyZ1 activity of strain BZd1 (838 U/mL) with dual-promoter Pr PHpaII . These pof genes , 29.spoVG was selected to use in this study (approximately 300 bp). However, it was much longer than that reported by Zuber et al. [spoVG, two truncated promoters PspoVG1 (120 bp) and PspoVG2 (180 bp) were cloned into plasmid pBHSS and resultant plasmids pBHSS1 (PspoVG-PspoVG1) and pBHSS2 (PspoVG-PspoVG2) were transformed further into B. subtilis WB600. The resulting recombinant strains BZd31 and BZd32 showed extracellular AmyZ1 activities of 1178.6 and 1232.3 U/mL , 36, and 48 h (p < 0.01), respectively , pBHSS4 (PspoVG-PspoVG142), pBHSS5 (PspoVG-PspoVG242), pBHSS6 (PspoVG42-PspoVG142), and pBHSS7 (PspoVG42-PspoVG242) were constructed based on PspoVG42, PspoVG1, and PspoVG2 and transformed into B. subtilis WB600 to obtain recombinant strains BZd33, BZd34, BZd35, BZd36, and BZd37, respectively. After 48 h of fermentation in shake flasks, the extracellular AmyZ1 activities of strains BZd33 to BZd37 were 1385, 1437.6, 1130.5, 1247.4, and 1344.6 U/mL, respectively . In addition, the AmyZ1 mRNA levels of strain BZd34 were 1.19-, 1.74-, 1.37-, and 1.62-fold higher than those of strain BZd31 at 12 (p < 0.001), 24, 36 (p < 0.001), and 48 h, respectively  mediated by promoter PNBP3510, obtained by changing the UP element, − 35, and − 10 regions of the promoter Pylb, was 26-fold higher than that mediated by the promoter Pylb and the highest transcription level of promoter PNBP3510 was 340-fold stronger than that of promoter Pylb [Notably, the strain BZd34 containing the promoter PB. subtilis. The resulting modified dual-promoters PspoVG-PspoVG2 (1232.3 U/mL), PspoVG-PspoVG42 (1385 U/mL), PspoVG-PspoVG142 (1437.6 U/mL), PspoVG42-PspoVG142 (1247.4 U/mL), and PspoVG42-PspoVG242 (1344.6 U/mL) mediated higher AmyZ1 activities than that mediated by original dual-promoter PspoVG-PspoVG (1139.8 U/mL) , indicating that this method may be an effective strategy to improve the strength of dual-promoter.In the previous study, the strategies of dual-promoter construction included combining different single promoters, changing the position of a promoter and so on , 36. In AmyZ1 mRNA secondary structure, the sequences of the first 11 codons of SPamyQ were changed according to the prediction results of UTR Designer  positively affected AmyZ1 expression  and BZd343 (optimized strain) were cultured in 3-L fermenters, respectively. The fed-batch strategy was used to obtain high cell density and high AmyZ1 activity in this study. During the fermentation process, the AmyZ1 activity in fermentation supernatants of strain BZ1 reached the maximum of 4097.8 U/mL at 36 h with productivity of 113.8 U/mL·h  and productivity (291.9 U/mL·h) of strain BZd343 in 3-L fermenter were 36- and 45.4-fold higher than those of strain B. amyloliquefaciens BAZ3-16 [B. subtilis, in order to meet the requirements of industrial applications better, follow-up strategies such as feed medium component or feed process optimization can be used to improve further the ability of the strain BZd343 to produce AmyZ1.In a previous study, Tang et al.  reported BAZ3-16 , respectB. subtilis. In this study, six single promoters were chose to evaluated their performances on AmyZ1 expression in shake-flask. The promoter PspoVG mediated the highest AmyZ1 activity (952.6 U/mL). Three dual-promoters were constructed and screened under identical conditions. The dual-promoter PspoVG-PspoVG showed the highest extracellular AmyZ1 activity (1139.8 U/mL). The modified promoter PspoVG-PspoVG142 further increased the AmyZ1 expression to 1437.6 U/mL. The AmyZ1 activities of the final recombinant strain BZd343 obtained by optimizing the 5'-proximal coding sequence were 1691.1 U/mL in shake flask culture and 14,012 U/mL in 3-L fermenter, respectively. It was the highest level of AmyZ1 secretory production in B. subtilis reported to date. This study thus provides an expression system with a potential for large-scale production of AmyZ1. This strategy might also be valuable for enhancing the production of other extracellular target proteins in B. subtilis.The strategies of promoter engineering and translation initiation efficiency optimization successfully enhanced the secretory expression of AmyZ1 in E. coli JM109 was used as a host strain for plasmids construction. B. subtilis WB600 was used as a host strain for AmyZ1 expression.All strains and plasmids used in this study are listed in Table B. subtilis shake flask fermentation. The seed culture was obtained by inoculating 5 mL of LB medium with 10 μL of bacterial solution from a glycerol tube cryopreserved at − 80 °C and incubated at 37 °C and 200 rpm for 10 h. The seed cultures  were then transferred to 250 mL shake flasks containing 100 mL 2 × YT medium. Next, these cultures were incubated at 30 °C and 200 rpm for 48 h. The medium used in 3-L fermenter was modified 2 × YT medium, which included 2.4% tryptone, 0.75% NaCl, 1.5% yeast extract, and 1% glycerol. The feeding solution consisted of 40% glycerol, 6% tryptone, and 2% yeast extract. The optical density of recombinant strain at 600 nm (OD600) was measured by spectrophotometer . 30 mg/L kanamycin or 100 mg/L ampicillin was added when needed and 10 mM CaCl2 was added for B. subtilis shake flask fermentation and 3-L fermenter fermentation.Luria–Bertani (LB) medium  was used for seed culture and 2 × YT medium  was used for The primers used in this study are listed in Additional file B. subtilis 168 and the Pontibacillus sp. ZY genomes were extracted using the rapid bacterial genomic DNA isolation kit . The Pontibacillus sp. ZY genome was used as the template for cloning AmyZ1 (GenBank accession No. MH325467.1) using polymerase chain reactions (PCR) with the primers P1/P2. After being digested with BamH I and Xba I, the AmyZ1 gene was then ligated into plasmid pHT43 [AmyZ1.The id pHT43  to genergroE was cloned from pHT43 using the primers P3/P4. The fragment of promoter P43 was cloned from pBEP43 [spoVG, PsecA, PodhA, and PlytR were cloned from B. subtilis 168 genome using the primers P7/P8, P9/P10, P11/P12, and P13/P14, respectively. One fragment derived from pHT43-AmyZ1 containing ribosome binding site, SPamyQ, and AmyZ1, was cloned using the primers P15/P16. Another fragment derived from pBEP43 containing B. subtilis ori and kan gene was cloned using the primers P17/P18. Then, these two fragments were ligated by overlap PCR to generate a backbone plasmid fragment. Finally, the various promoters and the backbone plasmid fragment were ligated using POE-PCR [groE), pBHP (P43), pBHS (PspoVG), pBHA (PsecA), pBHO (PodhA), and pBHL (PlytR), respectively.The plasmids containing different single promoters were constructed as follows. Firstly, the fragment of promoter Pm pBEP43  using th POE-PCR , generatgroE, P43, and PspoVG were cloned using the primers P3/P19, P5/P20, and P21/P22, respectively. Then, these three promoters were combined with PspoVG to generate dual-promoter PgroE-PspoVG, P43-PspoVG, and PspoVG-PspoVG, respectively. Finally, using the methods mentioned above, the three dual-promoters were ligated with backbone plasmid fragment to generate plasmids pBHES (PgroE-PspoVG), pBHPS (P43-PspoVG), and pBHSS (PspoVG-PspoVG), respectively.Based on corresponding templates, the upstream promoters PspoVG. Two truncated promoters, PspoVG1 and PspoVG2, were cloned from B. subtilis 168 genome using primers P23/P25 and P24/P26, respectively. They deleted a 180 bp and 120 bp sequence compared with the original promoter PspoVG, respectively. These two promoters were then combined with promoter PspoVG to generate plasmids pBHSS1 (PspoVG-PspoVG1) and pBHSS2 (PspoVG-PspoVG2), respectively. The promoter PspoVG42 was obtained by overlapping the two fragments cloned from B. subtilis 168 genome using primers P21/P28 and P27/P6, respectively. The promoter PspoVG was then combined with PspoVG42 to generate plasmid pBHSS3 (PspoVG-PspoVG42). Combining two strategies mentioned above, we constructed four plasmids containing different dual-promoters, named pBHSS4 (PspoVG-PspoVG142), pBHSS5 (PspoVG-PspoVG242), pBHSS6 (PspoVG42-PspoVG142), and pBHSS7 (PspoVG42-PspoVG242), respectively. All recombinant plasmids were transformed into B. subtilis WB600 following the method of Anagnostopoulos and Spizizen [The modified dual-promoters were constructed based on the plasmid pBHSS, including truncation and mutation of promoter PSpizizen .amyQ (designated as opt1 to opt5) were amplified from plasmid pBHSS4 using P29/P16, P30/P16, P31/P16, P32/P16, and P33/P16 as primers, respectively. The backbone plasmid fragment was amplified from plasmid pBHSS4 using P17/P34 as primers. Then, this backbone plasmid fragment and five optimization sequences were combined by POE-PCR to generate plasmids pBHSS8 (opt1), pBHSS9 (opt2), pBHSS10 (opt3), pBHSS11 (opt4), and pBHSS12 (opt5), respectively.The optimization sequences of the 5′-proximal coding sequence of SPThe activity of AmyZ1 was determined using the dinitrosalicylic acid (DNS) assay method as described by Fang et al. , in whicB. subtilis WB600 containing plasmid pBHE and then purified using the Ni-NAT system. The optimum temperature of AmyZ1 was determined at temperatures ranging from 20 to 70 °C. The optimum pH of AmyZ1 was assayed by measuring the activity in pH ranging from 5.0 to 10.0 at 40 °C using HAc-NaAc buffer , Na2HPO4-KH2PO4 buffer , Tris–HCl buffer , and Glycine–NaOH buffer , respectively. The highest activities of AmyZ1 were considered as 100% in the optimum temperature and optimum pH experiments, respectively. The thermostability of AmyZ1 was determined by incubating the AmyZ1 in Na2HPO4-KH2PO4 buffer  at temperatures ranging from 20 to 35 °C without Ca2+. The pH stability of AmyZ1 was determined by dispersing the AmyZ1 at pH 6.0, 6.5, and 7.0 without Ca2+, and the residual activities were measured at appropriate intervals. The initial activities of AmyZ1 were considered as 100% in the thermostability and pH stability experiments, respectively.The AmyZ1 was expressed in recombinant strain amyQ was predicted by UTR Designer (https://sbi.postech.ac.kr/utr_designer/) [http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). The five optimized sequences (designated as opt1 to opt5) were finally obtained after removing the sequences containing the hairpin at the first 13 nucleotide residue of sequences   using itThe supernatant samples were mixed with 2 × loading buffer. The resulting mixtures were then heated at 100 °C for 10 min and evaluated with 12% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R-250.AmyZ1 with the 16S rRNA gene as the reference. The AmyZ1 and 16S rRNA genes were amplified from the cDNA obtained above with primers P35/P36 and P37/P38, respectively. The reactions were carried out in the 96-well plates using SYBR Green Premix Pro Taq HS qPCR Kit  on a LightCycler 96 Real-Time PCR system  according to the manufacturer's protocols. The thermal cycling conditions contained an initial denaturation step (30 s at 95 °C); 50 cycles of denaturation (5 s at 95 °C); primer annealing and elongation (30 s at 60 °C); and a melt-curve step . The data was analyzed using the 2−ΔΔCt methodology [Total RNAs of samples were isolated using the Bacteria Total RNA Isolation Kit . Then, the cDNA was obtained using The Evo M-MLV RT Kit . The obtained cDNA was used as template for qPCR to measure the transcription level of gene hodology .4OH and HCl. After fermentation about 10 h, the initial glycerol was consumed. Then, the fed-batch cultivation started with a feeding speed of 0–18 g glycerol per hour to maintain a glycerol concentration of 0.2 to 1.0 g/L. Samples were withdrawn every 4 h and centrifuged at 12,000×g for 10 min at 4 °C to obtain the culture supernatants.The seed culture was grown in a 250 mL shake flask containing 60 mL 2 × YT medium supplemented with 30 mg/L kanamycin at 37 °C and 200 rpm for 12 h. 3-L Fermenter  containing 1.2 L fermentation medium was then inoculated with the seed culture . The dissolved oxygen tension was controlled by automatically adjusting the speed of the stirrer and airflow rate (2.0–5.0 L/min). The temperature was kept at 30 °C, and the pH was controlled at 7.0 using NHt-test and differences resulting in p < 0.05 was considered statistically significant.All experiments were conducted independently in triplicate. Data was presented as the average ± standard deviation. Statistical analysis was conducted using Student’s Additional file 1: Fig. S1 SDS-PAGE analysis of AmyZ1 purified using Ni-NAT system. Fig. S2 Effect of the 5'-proximal coding sequence optimization on the extracellular AmyZ1 expression. Table S1 Purification of AmyZ1. Table S2 Primers used in this study. Table S3 The sequences of different promoters used in this study.