For enhanced fuel cell performance at sub-optimal temperatures, the CMFA–ZnO heterostructure composite was developed. We have shown that a button-sized SOFC fueled by H2 and ambient air can provide 835 mW/cm2 of power and 2216 mA/cm2 of current at 550 °C, possibly functioning down to 450 °C. In addition, the oxygen vacancy formation energy and activation energy of the CMFA–ZnO heterostructure composite is lower than those of the individual CMFA and ZnO, facilitating ion transit. The improved ionic conduction of the CMFA–ZnO heterostructure composite was investigated using several transmission and spectroscopic measures, including X-ray diffraction, photoelectron, and UV–visible spectroscopy, and density functional theory (DFT) calculations. These findings suggest that the heterostructure approach is practical for LT-SOFCs.Improving the ionic conductivity and slow oxygen reduction electro-catalytic activity of reactions occurring at low operating temperature would do wonders for the widespread use of low-operating temperature ceramic fuel cells . In this work, we present a novel semiconductor heterostructure composite made of a spinel-like structure of Co The main concern is SOFCs (solid oxide fuel cells) converting the chemical energy of storage multi-fuels into electrical energy with zero pollution and remarkably better efficiency. Two main benefits of SOFCs include the usage of multi-fuels and flexibility in modularity for energy conversion technology, which has recently attracted immense attention ,2,3. HowGd0.1Ce0.O2 have b0.2TiO3) materials were used as an electrolyte for the application of ceramic fuel cells. It delivered high ionic conduction and an excellent fuel cell performance of 534 mW/cm2 at 520 °C. The Schottky junction phenomenon was proposed to suppress the electronic conduction and excel the ions [3 and SrTiO3 and attained maximum fuel cell performance at the lower temperatures of 500 and 550 °C [Recently, a new semiconductor-based fuel cell technology approach uses electrolytes and electrodes to replace conventional electrolytes and electrodes and mitigate the above-stated concerns. The semiconductor-based fuel cell uses semiconductor ionic materials (SIMs), which possess both ionic and electronic conduction while maintaining better and stable OCV operation with high performance. Most importantly, SIMs can operate at low operational temperatures without power loss and short-circuiting issues ,11,12. Ithe ions ,14. Simid 550 °C ,16. Mored 550 °C ,18,19,200.4Fe0.4Zr0.1Y0.1O3−δ (p-type semiconductor) as an electrolyte via compositing with ZnO (n-type semiconductor) to develop a p-n (BCFZY–ZnO) heterojunction. The constructed heterojunction delivered a peak power density of 643 mW/cm2, a better OCV of 1.01 V, and higher ionic conductivity of 0.21 S/cm at 550 °C [2 with an OCV of 1.06 V at 520 °C [3 (n-type) to shape into an SFT–WO3 p-n heterojunction which delivered an excellent power output of 875 mW/cm2 and high ionic conductivity of 0.2 S/cm at 520 °C [Moreover, the semiconductor ionic composite heterostructure approach was adopted at low operational temperatures to boost the ionic conduction and fuel cell performance. Xia et al. used triple conducting semiconductor perovskite BaCot 520 °C . Additiot 520 °C . In addit 520 °C ,24. Furtt 520 °C ,23,25,260.8Co0.15Al0.05LiO2−δ) to shape the fuel cell device in the following configuration: Ni-NCAL/CMFA–ZnO/NCAL-Ni. It delivered high ionic conductivity and appreciable power output at a low operating temperature of 450–550 °C. The attained high ionic conduction and better performance realized better functionality via constructing the p-n heterojunction, further suppressing the electronic conduction and promoting the ionic transport. Moreover, our investigation includes preparing and testing the feasibility of CMFA–ZnO electrolyte and performing different characterizations like XRD, SEM, HR-TEM + EDS mapping, UV–visible spectroscopy, and XPS. The attained results and new approach lead to insight from semiconductor and junction perspectives to the design of advanced electrolytes.Based on the above-stated literature on semiconductor technology and the advantages of composite heterostructure semiconductors, we have designed a new homogenous composite semiconductor p-n heterojunction of CMFA–ZnO as an electrolyte where CMFA is a p-type semiconductor while ZnO is an n-type semiconductor. The prepared composite heterostructure was well sandwiched between two symmetrical electrodes of Ni foam NCAL 2.6H2O, Mn (NO3)2.6H2O, Fe (NO3)2.6H2O, and Al (NO3)2.9H2O  were added into the homogenous solution. Finally, the solution was stirred at 80 degrees Celsius, 240 revolutions per minute (rpm), for another 6 h, and a brownish gel of CMFA was formed. The resulting brownish gel was then aged for 24 h at room temperature before being dried in an oven set at 150 degrees Celsius. Once dry, the gel was ground into a powder and calcined in the air at 850 degrees Celsius in a muffle furnace for 8 h.Spinel-structure sol-gel processing using citric acid and ethylenediaminetetraacetic acid (EDTA) as complexing agents yielded CMFA residues. Step one involved dissolving 0.1 moles of EDTA in de-ionized water and adding NHThe commercially purchased ZnO powder was used as a heterostructure component with CMFA powder with different molar ratios of 9:1, 8:2, 7:3, 6:4, and 5:5. In detail, both powders (ZnO and CMFA) were mixed in a motor pestle and ground well for 2 h. After obtaining both CMFA and ZnO powders, the mixtures were prepared via a solid-state mixing procedure by ball milling the synthesized CMFA and ZnO powders in different weight wt% ratios for 10 h at 400 rpm. Ethanol was used as a dispersing medium. The mixed powders were then sintered at 800 °C for 2 h and ground to obtain CMFA–ZnO.A Bruker D8 (X-ray diffractor meter) using Cu-Kα radiation (λ = 1.5418 Å) was used for measuring the XRD patterns of the ZnO, CMFA, and CMFA–ZnO heterostructure composite. The surface morphologies of ZnO, CMFA, and CMFA–ZnO were studied using a scanning electron microscopy of Merlin compacts (Zeiss). The Raman analyses of CMFA, ZnO, and CMFA–ZnO were performed using an NT-MDT (Russia) spectrometer with a 532 nm solid laser source. The surface properties of CMFA, ZnO, and CMFA–ZnO were studied using an X-ray photoelectron spectroscopy  with an Al Kα source Gamry Reference 3000 (pine instruments the U.S), and ZSIMPWIN software was used to measure and analyze the electrochemical impedance data in the open-circuit voltage condition in the 0.1 HZ to 1 MHz frequency range. A dc signal of 10 mV was applied to record the data.0.8Co0.15Al0.05LiO2−δ (NCAL from Bamo Sci. & Tech. Joint Stock Ltd.) electrodes. First, a slurry of the NCAL was made in terpinol and was brushed onto Ni-foam, followed by a desiccation process at 120 °C for 1 h. Afterwards, a dried piece of NCAL-painted Ni-foam was added in a steel dye, followed by CMFA, ZnO, or CMFA–ZnO electrolyte powders (0.20 g) and another piece of NCAL-painted Ni-foam. However, the three-layered structure of the cell was pressed unanimously at 200 MPa. The same protocol was applied to prepare all cells. Thus, in the end, we got different ceramic cells to measure the performance, such as Ni-foam NCAL/CMFA/Ni-NCAL, Ni-foam NCAL/ZnO/Ni-NCAL, and Ni-foam NCAL/CMFA–ZnO/Ni-NCAL for comparative study. The performance demonstration of a single CFC was done using 3% H2O-humidified hydrogen. The H2/air pressure was set to 100–120 ± 5% mL/min.The practical applicability of CMFA and its heterostructure composite with ZnO as an electrolyte in a ceramic fuel cell and the fabrication of CFC devices were arranged by dry pressing. As-prepared CMFA, ZnO, and CMFA–ZnO heterostructure composite electrolyte membranes were pressed between two symmetrical Nio is the vacancy formation energy, Etot (Voq) is the superlattice total energy in q charge state, and Etot  is the total energy of the ideal structure. Additionally, μ is the chemical potential and EF is the orientation of the Fermi level. Evalan refers to the valence band maximum and ∆V represents changes in the electrostatic potential with defective lattice.Spin-polarized DFT calculations on individual CMFA and CMFA–ZnO heterostructure composite were carried out using CASTEP  on Materials Studio 8.0. within the projector-augmented wave (PAW). The cubic spinel structure of CMFA (a = b = c = 0.06551 nm and α = β = γ = 60°) with space group Fm-3m-22-1086 and hexagonal structure ZnO with space group Fm-3m-225 and lattice parameters a = b = c = 5.4130 Å were used to optimize the crystal structures and their properties. The generalized gradient approximation and Perdew–Burke–Ernzerh (GGA + PBE) method were used to control the electron exchange correlation energy. Meanwhile, the LDA + U method was used with U = 4.5 eV for Fe-3d and U = 5.6 eV for Ce-4f. The energy cutoff for the plane-wave basis set was 720 eV, and the PAW data sets were used with the following valence electronic states: Fe , and O . The slab model was applied to construct the CMFA–ZnO heterostructure, and each slab of the CMFA surface and ZnO layer were separately computed in advance. A √2 × 3√2 × 1 supercell structure was created comprising (3 × 3 × 2) surface units for ZnO (111) and (2 × 2 × 2) surface units of CMFA (001) with minimized lattice mismatch <5%. A sufficient vacuum was applied to protect the self-interaction in the z-direction. The oxygen vacancy formation energy on CMFA, ZnO and CMFA–ZnO surface was calculated using ,28:EVo =Furthermore, Moreover, the crystallographic structure of the bulk heterostructure composite CMFA–ZnO was determined by HR-TEM, as presented in The SOFCs used single CMFA, ZnO, and CMFA–ZnO heterostructure composites as their electrolytes, and the performance of each was assessed between 450 and 550 °C. The acquired current–voltage (I–V) characteristics for the CMFA and ZnO electrolyte fuel cells, separately at 550 °C, are shown in 2 and 450 mW/cm2, respectively, with an open circuit voltage (OCV) of 1.02 V and 1.05 V. This was better than what has been reported for semiconductor electrolytes SNO and LCAO [At 550 °C, the maximum power density of the CMFA and ZnO electrolyte fuel cells was 272 mW/cmand LCAO ,32. A Scand LCAO . Additioand LCAO . Moreoveand LCAO ,23,26,352 and a good OCV at different operating temperatures of 550–450 °C. Additionally, these results show that the devices can work at low temperatures with power outputs of 235 mW/cm2 at 450 °C, as shown in More appealingly, the CMFA–ZnO electrolyte fuel cell performed much better than the CMFA fuel cell. It had a peak power density of 872, 692, 582, 389, and 235 mW/cm2/air atmosphere at 550 °C and 550–450 °C, as shown in 0-R1Q1-R2Q2) where, R0 is the ohmic resistance, R1 is the charge transfer resistance, while R2 is the mass transfer resistance and Q is the constant phase elements [0), charge transfer resistance (R1), and mass transfer resistance (R2). At 550–450 °C, the CMFA–ZnO cell had small ohmic resistances, but at lower temperatures, the polarization resistance was much lower. This means that the charge transfer and mass transfer happened quickly [0 using EIS spectra. Additionally, this low ohmic and polarization resistance was caused by the formation of heterojunctions, a space charge region, and a built-in electric field, all of which helped charge move through the grain boundary interface.To determine the electrochemical and electrical properties of CMFA, ZnO, and CMFA–ZnO heterostructure, impedance spectra of CMFA, ZnO, and CMFA–ZnO electrolyte-based fuel cells were measured in an Helements . The EIS quickly ,36,37,38−1 at 520 °C), and SiC–ZnO  [We also believe that the ionic conductivity of the proposed electrolyte CMFA–ZnO at 550–450 °C is essential to the current work. Hence, we employed the unusual approach to calculate this property. The ionic conductivity is evaluated and computed from the I–V curve in  550 °C) ,41,42. TFurthermore, the absorbance spectra were measured using the UV–visible absorbance spectra, as depicted in The conduction band (CB) levels were determined using the formula Eg = Vb + Vc in conjunction with bandgap and valence band measurements. The CMFA and ZnO have distinct energy band level parameters; when CMFA and ZnO particles come into contact to form hetero-phasic interfaces, their distinct Fermi energy levels will converge to a continuous state at the interface region. Thus, the CMFA and ZnO can produce a p-n bulk heterojunction at the particle level in the composite. To depict the junction effect on electrochemical enhancement (ionic promotion) and charge separation (electron-blocking) process, Furthermore, as shown in 3+-based materials as shown in 2+ 2p1/2 and Fe2+ 2p3/2 are at 709.2 and 722 eV, respectively. In contrast, Fe3+ 2p1/2 and Fe3+ 2p3/2 are located at 710.963 and 725.1 eV, respectively, in the Fe 2p spectra shown in 3+ 2p1/2 and Fe3+ 2p3/2 peaks, as well as a satellite peak at 717.9 eV, are likewise connected to the BEs of 712.96 and 725.31 eV, respectively [2+ and Fe3+), as shown in Furthermore, as shown in ectively ,27,47,48As shown in The DFT technique was used to conduct theoretical studies of CMFA, ZnO, and the CMFA–ZnO heterostructure composite to better understand their electrical characteristics and ion diffusion . Figure In addition, the electrical properties of a material can be revealed by measuring its energy for oxygen vacancy formation (EVo). Vacancy creation at various sites was used to determine the (EVo) for the CMFA, ZnO, and CMFA–ZnO heterostructure composite . ZnO and CMFA lattice sites hosted the (EVo) for a different position, with Zn Vo, Co Vo-Fe occurring in the CMFA–ZnO lattice and at the interface. At the CMFA–ZnO heterostructure contacts, the (EVo) was only 0.45 eV, as depicted in 2 at 550 °C) was seen in the constructed CMFA–ZnO electrolyte. Many variables contributed to the well-established interface between ZnO and CMFA, responsible for the superior fuel cell performance and high ionic conductivity of the CMFA–ZnO heterostructure composite. Combining theoretical and experimental methods, we examined the mechanism for the transport of high ions. Oxygen vacancies were discovered to be abundant at the CMFA/ZnO interface, allowing for easy ion migration that may increase the self-consistency of the CMFA–ZnO structure and manage the interface-level charge transit capacities for better ionic conduction. The findings show that this strategy can be used to create novel alternatives to existing materials for advanced ceramic fuel cells, where it can improve performance and ionic conduction while also introducing novel functionalities  and where it may find use in other relevant energy applications, such as photocatalytic water splitting.Enhancing ionic conduction at low temperatures in spinel and hexagonal structure-based ZnO–CMFA heterostructure composites was the focus of this work. Higher ionic and electrochemical performance (872 mW/cm


Marsupenaeus japonicus (MjDAD1) was identified and characterized. The protein encoded by MjDAD1 has the typical structure and functional domains of the G-protein coupled receptor family. MjDAD1 expression was significantly upregulated in the gills and hepatopancreas after low temperature stress. Moreover, double-stranded RNA-mediated silencing of MjDAD1 significantly changed the levels of protein kinases (PKA and PKC), second messengers (cyclic AMP (cAMP), cyclic cGMP, calmodulin, and diacyl glycerol), and G-protein effectors (adenylate cyclase and phospholipase C). Furthermore, MjDAD1 silencing increased the apoptosis rate of gill and hepatopancreas cells. Thus, following binding to their specific receptors, G-protein effectors are activated by MjDAD1, leading to DAD1-cAMP/PKA pathway-mediated regulation of caspase-dependent mitochondrial apoptosis. We suggest that MjDAD1 is indispensable for the environmental adaptation of M. japonicus.Dopamine receptors (DARs) are important transmembrane receptors responsible for receiving extracellular signals in the DAR-mediated signaling pathway, and are involved in a variety of physiological functions. Herein, the D1 DAR gene from  The diverse superfamily of GPCRs, comprising seven-transmembrane (TM) proteins, transduces external signals by binding to various ligands to initiate intracellular signaling cascades .GPCRs ha1 and DA5 comprise D1-like receptors, which activate adenylyl cyclase (AC). AC activation leads to increased intracellular cyclic adenosine monophosphate (cAMP) levels and cell metabolic regulation, such as changes to the functions of ion channel function and GPCR desensitization, culminating in the release of neurotransmitters [2–DA4 comprise D2-like receptors, whose coupled signal transduction pathway inhibits AC, thereby decreasing cAMP levels. In invertebrates, DARs are vital for physiology, particularly cell signaling, learning and memory, reproduction, processing visual stimuli, mood/feeding behavior regulation, and other metabolic processes [Dopamine receptors’ (DARs) downstream signal regulation affects the host defense against the external environment. However, the DAR synergistic regulation mechanisms are mostly unknown. There are five subtypes of DAR DA1−DA5), all of which are members of the G protein-coupled receptors (GPCRs) family. DARs are further divided into D1-like and D2-like receptors based on their pharmacological profiles, signaling mechanisms, and conserved structures . Subtype, all of rocesses . HoweverMarsupenaeus japonicus , is found throughout the Indo-West Pacific [M. japonicus is a popular seafood produced in significant quantities by fishing and aquaculture. Currently, M. japonicus is farmed in several European and Asian countries, among which the largest producer is China, producing 44,548 tons in 2021 [The economically important crustacean, kuruma shrimp  Pacific . M. japo in 2021 . Moreove in 2021 ,11; howeM. japonicus (MjDAD1) was identified, and its role in temperature regulation was explored. We further characterized its functions and possible regulatory mechanisms to gain a detailed picture of communication between the crustacean immune and neuroendocrine systems. We explored the possible regulatory mechanism of MjDAD1 in M. japonicus under low temperature stress. Furthermore, MjDAD1 was silenced to identify cold stress-related alterations in critical neuroendocrine system factors, and the mRNA levels of apoptosis-associated genes in gills and hepatopancreas were also assessed. The results partially revealed MjDAD1’s functions in M. japonicus, and provided a mechanistic explanation for crustacean low temperature resistance.Therefore, investigations of the immunity of shrimp must be very helpful to the healthy aquaculture of crustaceans. Herein, the DAD1 gene from MjDAD1 was cloned using RACE technology. Sequence feature analysis of the cDNA was performed using NCBI ORF finder software . The MjDAD1 cDNA (GenBank accession number MK287993) has a total length of 2253 bp, including an 88 bp 5′ untranslated region (UTR) region, an ORF of 1254 bp, and a 3′ UTR of 911 bp. The cDNA contains a typical tail signal, aataaa. The 3′ UTR region contains a 26 bp polyA sequence. Starting from an ATG start codon and ending with a TAA stop codon, the ORF encodes a putative protein of 417 amino acids  showed that MjDAD1 (The prediction results of the SMATR software (t MjDAD1  containsPenaeus monodon (50.46%)  . Overall(50.46%) .MjDAD1 mRNA expression in eight different M. japonicus tissues was assessed using qRT-PCR (MjDAD1 showed the highest expression in muscle (p < 0.05) and the lowest in hemocytes (p < 0.05). qRT-PCR . MjDAD1 MjDAD1 mRNA expression in gills (p < 0.05). MjDAD1 expression did not change significantly in the 22 °C and control groups (p > 0.05).in gills A and hepin gills B increasM. japonicus were determined (p < 0.05). In the 22 °C group, the AC activity in the gills and hepatopancreas was higher than that in the control group at 72 h (p < 0.05).The G protein effectors including AC and PLC in the gills and hepatopancreas of termined . Under 1termined  and hepatermined  significp < 0.05). In the 16 °C group, the PLC level in the gills increased during 3−24 h, reached the minimum value at 24 h, and returned to the control groups’ level at 72 h, while in the hepatopancreas, the PLC activity was higher than that in the control group at 24 h (p < 0.05). In the 22 °C group, the PLC level in the gills decreased at 3 h (p < 0.05), and returned to the control group level at 24 h, while in the hepatopancreas, the PLC activity was higher than that in the control group at 24 h (p < 0.05)Under low temperature stress at 10 °C, the PLC activity in the gills  and hepaM. japonicus (p < 0.05). Under low temperature stress, the cAMP level in the gills (p < 0.05). In the 10 °C group, the cAMP activity (p < 0.05); in the 22 °C group, the cAMP activity in the hepatopancreas was higher than that in the control group at 72 h (p < 0.05).As shown in he gills  was highactivity  in the hp < 0.05). Under 22 °C stress, DAG levels in the gills and hepatopancreas first decreased and then increased, reaching its highest point at 72 h (p < 0.05).Under 10 °C and 16 °C low temperature stress, the overall DAG levels in the gills  and hepap < 0.05). Under low temperature stress at 22 °C, the CaM levels in the gills and hepatopancreas gradually increased, reaching its highest level at 72 h (p < 0.05).Similar to DAG levels, the CaM levels in the gills  and hepap < 0.05), and then decreased to the control level at 72 h. Under 22 °C low temperature stress, the PKA levels in the gills gradually increased, reaching a peak at 72 h (p < 0.05). Under low temperature stress at 10 °C and 16 °C, the PKA levels in the hepatopancreas (p < 0.05); in the 22 °C group, the PKA levels in the hepatopancreas decreased at 24 h (p < 0.05), then increased to the control level at 72 h.Under low temperature stress at 10 °C and 16 °C, the PKA levels in the gills  first inpancreas  first inp < 0.05). In the 16 °C group, the PKC levels in the gills were higher than in the control group at 24 h (p < 0.05), then decreased to the control level at 72 h. The PKC levels in the gills of the 22 °C group showed no significant difference compared with the control during the whole experiment. Under low temperature stress of 10 °C and 16 °C, the PKC activity in the hepatopancreas (p < 0.05), while in the 22 °C group, the PKC activity of the hepatopancreas was higher than in the control group at 72 h (p < 0.05).Under 10 °C low temperature stress, the PKC levels in the gills  were sigpancreas  was highMjDAD1 under low temperature stress was assessed using dsRNA interference. After injecting dsRNA into M. japonicus, the mRNA expression level of MjDAD1 in gill and hepatopancreas was detected using qRT-PCR. In comparison with the NS group, MjDAD1 expression reached its lowest value at 14 h after the injection of dsRNA, decreasing by 66% and 60% in the gills and hepatopancreas, respectively . The PLC level .Moreover, the results showed that, in the gills of the 28 °C  at 48 h A. The PK at 48 h B. When Mhe gills C of the he gills D of the MjDAD1 was downregulated, the expression levels of Bcl2 expression in the gills (p53 (cas3 (MjDAD1 silencing (p < 0.05). We speculated that MjDAD1 had a negative regulatory effect on Bcl2 and a positive feedback regulation effect on p53 and casp3.RNA interference showed that, when he gills C and hephe gills D were allls  with DAD1 from Penaeus monodon. MjDAD1 contains seven transmembrane domains, corresponding to G protein coupled receptors, and MjDAD1 has high similarity with other marine invertebrates in the transmembrane domain, indicating that MjDAD1 has been highly conserved during the evolution process. These analyses indicated that we had successfully cloned the full length cDNA of MjDAD1 from M. japonicus.We isolated and characterized the full length cDNA sequence of MjDAD1 is highly expressed in the eyestalk, indicating its involvement in neuroendocrine and immune regulation of M. japonicus. Under low temperature stress, MjDAD1 mRNA levels increased markedly within 3 h, suggesting that MjDAD1 might participate in the cold stress response of M. japonicus. The expression level of the D2 receptor (DRD4) in Litopenaeus vannamei changed under ammonia nitrogen exposure, and the expression of dopamine receptors EsDAD1 and EsDAD2 in Eriocheir sinensis also changed with the decrease in salinity during the desalination process [The eyestalk is an important neuroendocrine regulatory organ in crustaceans.  process ,13. ThesM. japonicus showed an overall trend of increase and then decrease. Previous studies have shown that cAMP and DAG, as second messengers, affect specific immunity by regulating PKA and PKC activities [Callinectes sapidus, D1-like dopamine receptors increased the production of cAMP in the posterior gills [MjDAD1 silencing significantly decreased the levels of G protein effectors (PLC and AC), intracellular second messengers , and PKA, while the content of PKC increased significantly, which demonstrate that MjDAD1 can transduce its signal via cAMP generation and Ca2+ mobilization in response to cold stress. Thus, we deduced that the MjDAD1 signaling pathway exists in M. japonicus and MjDAD1 is an ortholog of the vertebrate receptors.Studies have shown that DARs, following binding to their membrane receptors, modulate shrimp immune defense by regulating intracellular signaling transduction pathways (nuclear transcription factors-protein kinases), ultimately affecting proteins related to immunity ,15,16. Dtivities ,22. In tor gills . In the MjDAD1 silencing on the endogenous apoptosis pathway after low temperature treatment. Mitochondrial apoptosis-related gene expressions, specifically the anti-apoptotic gene Bcl2, and a gene encoding an apoptotic protein downstream of the cascade, specifically casp3, were determined. Yin et al. [Litopenaeus vannamei. Herein, MjDAD1 silencing increased p53 and casp3 expression  significantly compared with those in the control; however, they returned to control levels at 48 h. This suggested that MjDAD1 is involved in the regulation of the shrimp caspase-dependent mitochondrial apoptosis pathway. Similar to our results, Pirger et al. [Dopaminergic signaling and cellular apoptosis are believed to involve mitochondrial dysfunction and oxidative stress induced by low temperature ,25. Our n et al.  found thr et al.  found thThe Guidelines for the Care and Use of Laboratory Animals in China were followed when carrying out all the experiments. The Institutional Animal Care and Use Committee (IACUC) of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences , approved the study .M. japonicus: body weight = 13.04 ± 0.88 g), which were allowed to acclimate for 7 days in cement tanks  containing filtered cycling aerated seawater . The shrimp received fresh clam meat twice daily during acclimation up to 24 h before their use.A commercial aquaculture market  provided healthy shrimp . The water temperature was controlled using an Artificial Climate Chamber . The control water temperature was 28.0 ± 0.5 °C. Each group comprised ten shrimps, and each condition was applied as three replicates. We collected gill and hepatopancreas tissues, which were snap frozen in liquid nitrogen and placed at −80 °C before experimentation.The TRIzol Reagent  was used to extract total RNA from shrimp muscle, eyestalk, stomach, brain, thymus, gills, hepatopancreas, and hemocytes according to the supplier’s protocol. In addition, 1% agarose gel electrophoresis was used to determine the RNA quality and the RNA concentration was determine using a Nanodrop 2000 instrument . cDNA was synthesized from high quality RNA from each tissue.M. japonicus transcript database [MjDAD1 cDNA sequences. A 5′ and 3′ rapid amplification of cDNA ends (RACE) kit  was used to amplify further MjDAD1 cDNA sequences following the supplier’s guidelines. The RACE experiments used specific primers  database . The SMART program  was used to predict functional sites and domains in the putative MjDAD1 sequence. The molecular weight and isoelectric point (pI) were predicted using the ProtParam tool at the ExPASy molecular biology server . The MjDAD1 protein sequence was multiply aligned with other known DAD1 proteins using the CLUSTALW program package in the DNAMAN 8.0 software [The deduced protein sequence of software . The physoftware  in MEGA software  with supACTB (encoding β-actin)  using a SYBR Premix Ex Taq II kit (Takara). The reactions comprised 5 μL of SYBR Premix Ex Taq II (2×), 2 μL of cDNA, 0.4 μL of specific primers (10 mol/L), 0.2 μL of ROX Reference Dye II (50×), and 3.4 μL of RNase-free water. The cycling comprised initial denaturation at 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and 95 °C for 15 s; then 1 min at 60 °C and 15 s at 95 °C. Each sample was analyzed repeatedly to ensure that the data were reliable. The 2−∆∆Ct method [MjDAD1.We extracted RNA from tissues and used a PrimeScript RT Reagent Kit with gDNA Eraser (Takara) to obtain cDNA via reverse transcription. The open reading frames (ORFs) of the genes were used to design PCR primers to detect their expression levels; the control was β-actin) . The relt method  was emplThis study conducted temperature experiments at 10 °C and 28 °C gradients after siRNA interference. Each temperature gradient was divided into two groups, namely the RNAi group and the negative control (NC) treatment group. Shrimp were evenly divided into groups according to their respective amounts, and siRNA was injected at a rate of 1 µg/g at their fourth tail segment. The control group was injected with an NC negative control (meaningless double-stranded RNA), and gill and hepatopancreas tissues were collected from nine shrimp and stored in liquid nitrogen for RNA extraction at 3, 12, and 48 h (which were injected again at 24 h).Gill and hepatopancreas samples for histological examination were prepared according to previously published methods ,35. In bGill tissues were subjected to terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL) staining employing an In Situ Cell Death Detection Kit  following the supplier’s guidelines. In summary, the cell sections were deparaffinized, rehydrated, and digested with proteinase K for 30 min. The sections were added to the TUNEL reaction mixture and incubated in a humidified chamber at 37 °C for 1 h. Subsequently, the sections were rinsed using phosphate-buffered saline (PBS), stained using 3,3′-diaminobenzidine (DAB), subjected to Mayer-hematoxylin counterstaining, viewed under a microscope, and photographed. Image-Pro Plus 6.0  was used to quantitatively analyze the staining intensity.v:v) glycerol, 1.0 mM dithiothreitol, and 1.5 mM EDTA. The debris was removed by centrifugation at 12,000× g . The supernatants were further centrifuged at 3000× g  and the obtained supernatants were analyzed for their concentrations of PKC, PKA, CaM, DAG, cAMP, PLC, and AC, and their protein contents.Gill and hepatopancreas samples (0.1 g) were homogenized in buffer at 0 °C, composed of 20 mM Tris-HCl (pH 7.6), 10% , shrimp PLC (BP-E94030), shrimp cAMP (BPE94058), shrimp CaM (BPE94030), shrimp DAG (BPE94095), shrimp PKA (BPE94011), and shrimp PKC (BPE94012) ELISA kits . Their concentrations were determined following the supplier’s guidelines.Mjp53 and ACTB . The reaction conditions comprised: 10 min at 95 °C; 40 cycles of 95 °C for 30 s and 60 °C 34 s; 95 °C for 5 s; 60 °C for 1 min; and 95 °C for 15 s. The 2−∆∆Ct method [Trizol was used to extract RNA from the samples following the supplier’s protocol . HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit  was used to produce single-stranded cDNAs from the RNA, which were used as templates for the quantitative real-time PCR (qPCR) step of the qRT-PCR protocol. An Applied Biosystems™ 7500 Real-Time PCR instrument  with ChamQ SYBR qPCR Master Mix (High ROX Premixed) kit (Vazyme) were used to carry out the qPCR reactions. t method  was emplp value less than 0.05 was considered to indicate statistical significance. SPSS 23.0 for Windows  was employed for all statistical tests.Data are shown as the mean ± standard deviation (SD) of three independent experiments. Significant differences between control and experimental individuals were determined using one-way analysis of variance (ANOVA), followed by Dunnett’s test. A M. japonicus DAR gene, MjDAD1, whose expression was upregulated markedly by low temperature stress. MjDAD1 effects DA signaling, which alters PLC and AC levels, resulting in CaM, DAG, and cAMP concentration changes. Silencing of MjDAD1 resulted in downregulated Bcl2 expression in the hepatopancreas. Likewise, silencing of MjDAD1 led to low temperature-induced activation of apoptosis signaling pathway-associated genes. Ultimately, PKA/PKC induced changes to nuclear transcription factors, which regulated apoptosis. These results implied that D1-like receptors, via their positive association with the cAMP-PKA-CREB signaling pathway, have synergistic functions in the M. japonicus neuroendocrine immune (NEI) system.In summary, we cloned an


Lysine crotonylation (Kcr) is up-regulation in colorectal cancer (CRC) tissues, while its specific contribution remains uncertain. This study aimed to elucidate the role and mechanism of crotonylation on Lys27 of histone H3 (H3K27cr) in facilitating CRC metastasis.Immunohistochemistry was employed to investigate the correlation between H3K27cr and CRC metastasis. Both in vitro and in vivo assays employing loss function or gain function approaches were conducted to elucidate the role of LINC00922 in promoting CRC metastasis. ScRNA-seq analysis and immunoprecipitation analyses were employed to explore the underlying mechanism by which LINC00922 facilitates CRC metastasis through H3K27cr.Clinically, H3K27cr was upregulated in metastatic CRC tissues and positively correlated with advanced clinical stages. Functionally, knockdown of LINC00922 inhibited migration of CRC cells both in vitro and in vivo. Furthermore, the supplementation of NaCr restored the migration and invasion levels of LINC00922 stable knockdown cells by restoring the H3K27cr level. Mechanistically, LINC00922 promoted invasion and migration through H3K27cr mediated cell adhesion molecules (CAMs) in epithelial cells. Notably, LINC00922 interacted with the protein sirtuin 3 (SIRT3) and obstructed its binding to the promoter region of ETS1, leading to an elevation in the level of H3K27cr in this promoter region and the subsequent activation of ETS1 transcription.Our findings uncovered a novel regulatory function of H3K27cr, regulated by LINC00922, in facilitating CRC metastasis. This discovery contributed to a deeper comprehension of the involvement of histone crotonylation in the metastatic process of CRC.The online version contains supplementary material available at 10.1186/s12943-023-01859-y. A recently identified post-translational modification, known as lysine crotonylation (Kcr), has been observed in both human and mouse cells . Kcr hasCrotonyl-CoA can release from mitochondria and then translocate into nuclei, where it triggers histone crotonylation. Xu et al. revealed that BRD4 promotes the migration and invasion of prostate cancer (PCa) cells through histone crotonylation . HoweverModification of histone crotonylation is a reversible thermodynamic process regulated by crotonyltransferases and decrotonylases. SIRT3 is predominantly localized in the mitochondrial matrix and is generally thought to be a NAD-dependent deacetylase. Recently, Wei et al. showed that treating cells with NAM, a SIRT3 inhibitor, for 24 h increases histone crotonylation level . AdditioStudies have revealed that long noncoding RNA (lncRNA) binds and recruits crotonylation-modifying enzymes to specific genome loci. For instance, lncRNA EPB41L4A-AS1 interacts with GCN5 and promotes its translocation to promoters of glucose metabolism-related genes . AdditioLncRNA LINC00922 promotes the proliferation, migration, and invasion of colorectal, lung, gastric, ovarian, and liver cancer cells. Several studies have suggested that LINC00922 not only functions as a microRNA sponge, but also plays a role in promoting cancer metastasis by recruiting DNA methyltransferases (DNMTs) to the promoter regions of metastatic genes, resulting in altered expression of these genes , 20. Our2 in a humidified incubator.Human CRC cell lines, including HCT116 (TCHu 99), LoVo (TCHu 82), DLD-1 (TCHu134), SW620 (TCHu101), and SW480 (SCSP-5033), were purchased from the cell bank of the Chinese academy of science . Human normal colorectal epithelial cell line FHC (CRL-1831) was purchased from American type culture collection . HCT116, LoVo, DLD-1, SW480, and FHC were cultured in Dulbecco’s modified eagle medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1% antibiotics . SW620 was cultured in RPMI 1640 medium  supplemented with 10% FBS and 1% antibiotics. All cells were cultivated at 37 °C under 5% COThe siRNAs were synthesized by integrated biotech solutions . The plasmids were purchased from Hebio  or YouBio . The transfection of siRNAs and plasmids was performed in a transient manner, according to the manufacturer’s instructions, using siLentFect lipid reagent  and liposomal transfection reagent , respectively. The sh-LINC00922 plasmid was constructed and then packaged into lentivirus by GenePharma . To obtain LINC00922 stable knockdown cell line, HCT116 cells were infected with lentivirus and successfully transfected cells were screened using 4 µg/mL puromycin  for two weeks. The sequences of siRNAs are shown in Table https://xena.ucsc.edu) and gene expression omnibus  database. To compare the levels of LINC00922 and H3K27cr between two groups of samples, the Mann-Whitney U test was employed. The levels of H3K27cr and LINC00922 across multiple sample groups were analyzed using one-way analysis of variance (ANOVA) analysis. Kaplan-Meier analysis was performed to obtain survival analysis. A chi-square test was performed to analyze the distribution of LINC00922 between cytosol and nucleus. The expression correlation between LINC00922 and ETS1 in CRC tissues was examined using the Pearson correlation test. KEGG was used to investigate the biological pathways. Meta-analysis was performed to study the association between LINC00922 and CRC metastasis.All the original clinical data and genomics information were downloaded from UCSC Xena (GSEA was performed using the fgsea package (version 1.16.0) in R . The CRCP < 0.05 were displayed using heatmap.The lncRNAs differently expressed between normal and CRC tissues were analyzed using edgeR package. Briefly, gene expression profile was downloaded from UCSC Xena database. The samples were divided into two groups according to normal or cancer. The lncRNAs with |fold-change| ≥2 and The single-cell transcriptomes of all samples from GSE196964 and GSE221575, four primary CRC tissues and two liver metastatic CRC tissues (GSM7058756 to GSM7058761) from GSE225857 were downloaded from GEO database. Following, data were analyzed using Seurat, presto, dplyr, msigdbr, tibble package in R. To remove the batch effects, the FindIntegrationAnchors function was employed to calculate the anchors, subsequently integrating all samples using the IntegrateData function. The marker of each cell-type was analyzed using FindAllMarkers function and then the cell-type was identified using CellMarker 2.0 database. The enrichment between gene sets and specific cell-type was analyzed using fgsea package and the results were displayed using ggplot2 package. The R code referred to the report of Wu et al. . We alsoCells were seeded into chambers containing 8 μm pores  with or without Matrigel  coating. Briefly, 200 µL serum-free medium suspended with cells and 600 µL medium supplemented with 10% FBS were added to the upper and lower chambers in a bicameral culture system, respectively. After 24 and 48 h, cells penetrating the pores were fixed with 4% paraformaldehyde solution and stained with 0.1% crystal violet. Subsequently, cells were observed under an Olympus microscope at a magnification of × 100. Finally, the pictures captured were analyzed using Image J software.LoVo cells were seeded into a 6-well plate and cultured to 95% confluence. A 200 µL pipette tip was used to scratch on cell monolayers. Following the removal of cellular debris by washing, the cells in the plate were cultured with a new medium that was devoid of serum. Photomicrographs were obtained with a Nikon digital camera  at ×100 magnification at 0 and 48 h. Lastly, the change in the wound size was measured.Total protein was harvested from cells using cell lysis buffer (50 mM Tris-HCl (pH 8.0), 4 M urea, and 1% Triton X-100) supplemented with protease inhibitors. To determine the concentration of cell lysates, the BCA kit  was employed. Subsequently, proteins of varying molecular weights were separated through SDS-PAGE and transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked with 5% skim milk for 2 h and then incubated with primary antibodies overnight at 4 ℃, followed by secondary antibodies for 1 h. The following antibodies were used: anti-ETS1 , anti-E-Cad , anti-Vimentin , anti-Snail1 , anti-SIRT3 , anti-H3K27cr , anti-GAPDH , anti-H3 , anti-rabbit IgG , and anti-mouse IgG . The intensity of bands was scanned and then measured using Image J software.−ΔΔCt formulae. Primers used for qPCR are listed in Table Total RNA from tissues and cells was extracted using TRIzol reagent. The RNA was then reverse transcribed into cDNA using SweScript RT I First Strand cDNA Synthesis Kit . QRT-PCR was performed using 2×SYBR Green qPCR Master Mix (High ROX) . The qPCR process was performed in triplicate, and the relative fold-change of genes was normalized using GAPDH and calculated using the 2The immunofluorescence assay was conducted using antibodies against ETS1 (1:50), H3K27cr (1:100), and SIRT3 (1:50). The experiment began with the seeding of cells onto coverslips, followed by a 24 h growth period. Afterwards, the cells were washed three times with PBS and fixed with cold methanol for 5 min. Then, the samples were blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with primary antibody for 2 h at room temperature. Subsequently, the glasses were immersed into 3% FBS containing Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG(H + L)  and/or Alexa Fluor 488-conjugated Affinipure Goat Anti-Mouse IgG(H + L)  for 1 h at room temperature. Finally, the cells were stained with DAPI and visualized using a fluorescence microscope or STELLARIS 5 confocal fluorescence microscope (Leica).Cells were cross-linked with 1% formaldehyde and then re-suspended in ChIP lysis buffer (50 mM Tris-HCl (PH = 8.0), 5 mM EDTA, 0.1% deoxycholate, 1% Triton X-100, 150 mM NaCl and 20 µL/mL PIC). After 10 min, cells were sonicated and centrifuged. The supernatants were subsequently incubated with antibodies and Protein A/G beads for 4 h. In the ChIP assay, anti-H3K27cr and anti-SIRT3 antibodies were employed, while rabbit IgG  and mouse IgG  served as a control. This was followed by washing of the complex samples thrice to obtain clean DNA samples. Finally, DNA samples were analyzed using qRT-PCR assay or sequenced by Orizymes company .RNA in the cytosol and nucleus was extracted using nuclear and cytoplasmic protein extraction kit . Firstly, PMSF and RRI were added into cytoplasmic protein extraction reagent A. Then, cells were lysed with pre-cooling reagents A and B on ice for 15 min. Subsequently, cell lysate was centrifuged, and the supernatant was collected to extract cytoplasmic RNA. The sediment was resuspended in reagent A and reagent B while kept on ice for 45 min, and subsequently washed thrice with PBS to remove any remaining cytoplasm. Finally, Trizol was used to extract nuclear RNA from the centrifuged deposit.2, 10 mM HEPES (PH = 7.0), 0.5% NP-40, 1 mM DTT, 100 U/mL RRI, 20 µL/mL protease inhibitor and 2 mM vanadyl ribonucleotide complex solution). Subsequently, the cell lysate was mixed with 3 µg of IP-grade anti-SIRT3 and left to incubate overnight at 4 °C, and mouse IgG antibodies served as a control. The reaction mixture was then supplemented with 40 µL of Protein A/G beads and left to react for 4 h. The resulting precipitated RNA was extracted, purified and subjected to qRT-PCR analysis.Cells were harvested and then digested using RIP lysis buffer . Briefly, the cells were spread on coverslips in a 12-well plate. The following day, the coverslips were washed three times with PBS, and the cells were permeabilized with CSK buffer on ice for 5 min and then fixed with 4% paraformaldehyde for 10 min. Then, cells were successively dehydrated with 80, 95, and 100% ethanol for 3 min at each concentration and incubated with the LINC00922 probe at 37 °C overnight in a dark and humid chamber. The coverslips were then washed three times in freshly-prepared 50% formamide and 2 × SSC buffer at 42 °C for 5 min. Subsequently, the cells were blocked with 1% BSA for 15 min and then exposed to SIRT3 antibody (1:50) for 45 min. Finally, the specimens were counterstained with DAPI and mounted to be examined on a STELLARIS 5 confocal fluorescence microscope (Leica).Tumor tissues were purchased from Shanghai Outdo Biotech company , including colorectal tumor samples from 45 patients and adjacent tissues from 14 patients. The 45 tumor samples in contained 10 distant metastatic colorectal cancer tissues, 16 primary colorectal cancer tissues, 11 colorectal adenoma tissues, and 8 positive lymph node tissues. Tissues were incubated twice in xylene for 15 min each time and then in 100% ethanol twice for 7 min each time, and once in 90, 80, and 70% ethanol for 5 min each. Afterward, 0.01 M citrate buffer was used for antigen retrieval and then 3% hydrogen peroxide was used to quench peroxidase activity. Tissues were blocked with BSA, then incubated with anti-H3K27cr antibody (1:100) at 4 °C overnight, and then incubated with the secondary antibody labeled with HRP  for 30 min at room temperature. Next, the slices were dehydrated in succession, sealed with neutral resin, and scrutinized under a microscope. Ultimately, slices were evaluated independently and blindly by two observers.The levels of H3K27cr were quantified using histochemistry score (H-score), with multiplication of the staining intensity with the positive cell area. Staining intensity was evaluated using a grade system ranging from 0 to 3, where 0 indicating no staining, 1 indicating weak staining, 2 indicating moderate staining, and 3 indicating strong staining. The positive cell area was measured as a percentage ranging from 0 to 100%. As a result, the H3K27cr levels spanned from 0 to 300%. In Fig. 6 HCT116 cells, with or without stable LINC00922 knockdown, were injected into each mouse via the tail vein. Four mice injected with control cells died during animal experiment. After five weeks, four and eight mice in the control and sh-LINC00922 groups were killed, respectively. The lungs of mice with tumor implant nodules were collected, photographed, and statistically analyzed.To explore the effect of LINC00922 in the CRC metastasis, nude mice were used to construct a lung metastasis model. The BALB/c nude mice were developed at GemPharmatech  and were randomly divided into two groups (n = 8/group). A sum of 3 × 10t-test, Mann-Whitney U test, or Chi-square test. Differences among several groups were evaluated using one-way ANOVA. Association between the two groups was evaluated using Pearson’s correlation analyses. P < 0.05 was considered statistically significant.All experiments were performed in independent triplicates, and the data were presented as mean ± SD. Statistical analysis was performed using SPSS software version 22.0, and graphs were plotted using GraphPad Prism software version 8.0. Differences between the two groups were evaluated using an unpaired two-tailed Student’s We conducted an investigation into the level of H3K27cr in colorectal tumor samples obtained from 45 patients, as well as adjacent tissues from 14 patients, utilizing immunohistochemical staining. Intriguingly, the findings demonstrated a significant elevation in H3K27cr level at distant metastasis sites )  between LINC00922 overexpressed and control cells , contained more than 5 genes Fig. . In the Previous studies indicate that ETS1 regulates the expression of CAMs. Therefore, we next investigated whether LINC00922 regulated invasion and migration via ETS1. Firstly, this study demonstrated that exogenous ETS1 promoted the invasion, migration, and cell motility of CRC cells as well Fig. A-B. AddiNext, we investigated whether LINC00922 altered ETS1 expression. We conducted the qRT-PCR analysis in CRC tissues from 28 patients. The result exhibited a direct correlation between the expression of LINC00922 and ETS1 Fig. A. KnockdWhether LINC00922 regulated ETS1 expression via H3K27cr was investigated. Firstly, HCT116 cells treated with 10 mM NaCr increased the levels of H3K27cr and ETS1, indicating H3K27cr might activate ETS1 transcription Fig. . The ChIHow LINC00922 altered the enrichment level of H3K27cr on the ETS1 promoter was studied. ChIP-seq of HCT116 cells revealed that SIRT3 was distributed near the TSS region Fig. . HoweverClinical evidence shows variation in Kcr level in different tumors. Previous studies revealed up-regulated Kcr level in cancers, including colon, prostate, thyroid, esophagus, pancreas, and lung neoplasms, and down-regulated Kcr level in cancers, including liver, stomach, and kidney carcinomas , 25. ZhaThe association between Kcr and cancer metastasis is still poorly understood. Wan et al. discovered a correlation between Kcr level and TNM stage of hepatocellular carcinoma (HCC) and increased Kcr level inhibited HCC cells motility . In contMost prior investigations have concentrated on the deacetylase activity of SIRT3 rather than its decrotonylase activity. SIRT3 is capable of deacetylating a wide range of substrates, including both histone and non-histone proteins. Multiple studies have proposed the potential recruitment of SIRT3 to specific genomic locations. For instance, Toshinor and colleagues discovered that SIRT3 could regulate gene expression through chromatin localization and interaction with chromatin in defined regions . SIRT3 wOur study presents a working model for LINC00922-mediated SIRT3 recruitment and H3K27cr modification in human cancer metastasis. Gaining a better understanding of the critical role of histone crotonylation in biological processes could potentially lead to novel cancer treatment strategies.Below is the link to the electronic supplementary material.Additional file 1: Fig. S1 to Fig. S5Additional file 2: Table S3Additional file 3: Table S4Additional file 4: Table S5Additional file 5: Table S6Additional file 6: Supplementary Video 1


The conventional Metal cutting fluids (MCFs) used are mineral-based petroleum oils that perform well but are toxic and difficult to dispose of; therefore, these are hazardous to human health as well as the environment. This issue can be solved by using natural vegetable oil-based MCF, which are readily available, environment and human-friendly, and renewable. Therefore, we synthesized various types of emulsifiers  based on recycled vegetable oil (RO) from spent bleaching earth (SBE), and elucidated their chemical structures by different spectroscopic techniques. The individually synthesized emulsifiers  at different ratios (8–15 by wt.%) and mixed emulsifiers  at different ratios (8–12 by wt.%) were utilized as additives in the preparation of different vegetable residual oil-based MCF formulations. The mixed emulsifiers at different ratios of nonionic/nonionic with hydrophilic-lipophilic balance (HLB) value 10 , and anionic/nonionic  exhibited stable emulsions compared to individual emulsifiers. Formulas (I and VI) displayed good protection effectiveness in corrosion tests. Formula VI had better wettability  and a smaller particle size (63.97 nm). Tribological properties of Formula VI were also performed. The results exhibit that Formula VI is consistent with the commercial sample. As a result, this study contributed to the resolution of one of the industry's problems Also, it absorbs varying amounts of edible oils according to the type of oil and the technologies used. Spent bleaching earth (SBE) is a waste oil refinery having a high proportion of vegetable oil (~ 20–40%)5. Significant volumes of SBE are discarded in landfills, which cause fire and pollution risks owing to the large oil content in the earth which leads to climate changes7. SBE oil would have been wasted if it had not been retrieved which would have caused oil depletion in the long term. Thus, oil preserved in SBE can be recycled and reused in industrial applications as raw materials in order to reduce costs in the oil refining industry14. However, The recycled vegetable oil would have high free fatty acids and peroxide value, and would not be suitable for food application12. Recently, the application of vegetable oil-based metal cutting fluids (MCFs) has been proposed. MCFs are an essential aspect of the modern industrial system. During the metal machining process; a high temperature at the cutting zone is produced by the friction between the cutting tool-work piece and cutting tool-chip interfaces. This heat causes a shorter lifetime of the tool, a higher roughness of the surface, and decreases the dimensional sensitivity of the work material. This finding is more significant when machining difficult-to-cut materials owing to higher heat occurrence15. One of the methods to reduce the heat generated at the tool-workpiece interface is the use of MCFs in machining operations15. As a result, the current demand for MCFs is increasing16 and the development of new MCFs with superior performance based on waste products is required17. MCFs are used during the machining operations to provide lubrication and cooling effects between the cutting tool-work piece and the cutting tool-chip. Hence, the heat will be carried away by the lubricant during the cutting process21. Also, they facilitate chip flushing away from the machining zone and prevent adhesion of the workpiece to the cutting tool.In the edible oil refining industry, bleaching earth (BE) is utilized to eliminate color, residual gums, phospholipids, and metals from the oil22. The machining parameters improved utilizing minimum quantity lubrication are cutting temperature, surface roughness, tool wear, and cutting forces achieving good machinability of metal23.High temperature is the primary cause of tool wear in dry cutting, resulting in tool failure. As a result, the presence of coolant is critical in machining. Cooling techniques used in machining include conventional cooling, cryogenic cooling, minimum quantity lubrication (MQL), nano-fluid machining, and air cooling. MQL; most typically used in metal cutting operations: delivers a combined action of coolant and lubricant, which reduces setup and fluid costs. MQL fluid evaporates at the cutting zone, eliminating the need for cutting fluid maintenance, circulation, and disposal25. Toxic and corrosive slurries and fluids are employed in cutting and polishing fluids. This induces pollution in the environment. To solve this challenge, novel green fluids are proposed31. Using the novel green fluids, high-performance surfaces are manufactured for use in semiconductor, optoelectronics, and aerospace industries32. These novel green fluids are a great contribution to conventional manufacturing, and the most important is reducing the pollution to the environment effectively33.The key ingredient of industrial metal cutting fluids is mineral oil and other additives with distinctive properties. Mineral oil-based MCFs are non-biodegradable and have a negative impact on the environment. Furthermore, these MCFs pose numerous health risks24. It would be very appealing to replace petroleum-based MCFs with bio-based MCFs (vegetable oil-based MCFs), which are environmentally friendly, renewable, less toxic, and highly biodegradable. Consequently, in addition to having superior coolant and lubricant properties, high-quality MCFs should be safe to discard after use36.Therefore, the formulation of metal cutting fluid dependent on vegetable oils as a renewable source is regarded as one of the industry's unique solutionsAs a result, this research focuses on optimizing the benefits of recycled vegetable oil as a base ingredient for the preparation of various types of surface active agents. The synthesized surfactants are evaluated as emulsifiers, corrosion inhibitors, and biocides and they’re applied in designing various metal-cutting fluid formulations. Thus, the synthesized surfactants fulfill all the requirements of cutting fluid formulations. Additionally, recycled vegetable oil is used as a vegetable oil base in metal-cutting fluid formulations.In this study, residual vegetable oil recovery was extracted from SBE and chemically modified into multifunctional surfactant compounds which were utilized as additives in the preparation of different vegetable oil-based metal-cutting fluid formulations. In addition, the role of individual and mixed emulsifiers at different ratios in the stabilization of the formulations was studied. These formulations were evaluated using oil stability, emulsion stability; corrosion inhibition, wettability, particle size, and wear resistance tests and then compared these data with commercial sample results.Spent bleaching earth (SBE) was provided by Tanta Oil and Soap Company. Soybean oil accounts for 75% of domestic oil production. Consequently, the SBE sample used is taken from refining soybean oil.14 as shown in Fig. Cold extraction with n-hexane solvent was used to extract the oil from SBE. One liter of n-hexane was put into a 2L bottle holding 0.5 kg of fresh SBE. The bottle was tightly closed and set aside for 72 h at room temperature. Afterward, the resulting solution was decanted and filtered. The solution (oil + n-hexane) was then concentrated by distillation utilizing a vacuum pump to collect the recycled vegetable oil. From the 0.5 kg SBE, 55 g of oil was collected, producing an oil yield of 11%. The solvent that was recovered might be used againPhysicochemical properties of the recycled vegetable oil such as density, color, viscosity, saponification value, free fatty acid, total acid value, iodine value, peroxide value, flash point, molecular weight, and fatty acid composition were reported in Table 37 with HP 6890 plus gas chromatography .The recycled vegetable oil's fatty acid composition was identified via a modified methodThe synthesis of anionic emulsifier was illustrated in Fig. 2SO4 to obtain yellow pasty N-alkyl ethanolamine N-(2-hydroxyethyl) alkyl amide (HEA-A) or N-(2-aminoethyl) alkyl amide (AEAA)38.In a three-neck round-bottom flask, 0.1 mol RO, monoethanolamine or ethylenediamine (0.31 mol), and 0.2 wt.% anhydrous sodium acetate was agitated for 3 h at 160 °C. The reaction product was dissolved in dichloromethane, washed three times with distilled water, and dried over anhydrous Na40.N-(2-hydroxyethyl) alkyl amide  was dissolved in 20 ml chloroform at 5 °C, chlorosulfonic acid  was added dropwise slowly for 6 min, swirling the mixture magnetically. At the same temperature, stirring was continued for an additional three hours. n-Butanol was used to dilute the contents, and 18 N aqueous sodium hydroxide solution was used to neutralize them. The mixture of water and n-butanol was separated, and the residual material was dried at 80 °C under reduced pressure to obtain a pale brown waxy productThe synthesis of nonionic emulsifiers was illustrated in Fig. 41.N-(2-aminoethyl) alkyl amide AEAA , succinic anhydride  were dissolved in 1,4-dioxane and agitated for 15 h at 80 °C. The solvent was removed under reduced pressure and the subsequent product was washed with diethyl ether, filtered off as a pale yellow solid with m.p. 105–110 °C2SO4, and concentrated under reduced pressure. The four nonionic emulsifiers (NS1-4) were obtained as viscous liquids42.In 100 mL round bottom flask was charged with 4-amino)-4-oxobutanoic acid , polyethylene glycol with different molecular weight  (33.26 mmol), and p-toluene sulfonic acid . Toluene was added as a solvent, and the mixture was refluxed for 8 h using a Dean-Stark apparatus. After cooling to ambient temperature, the mixture was treated with dichloromethane, then three times washed with water, dried over anhydrous NaThe synthesis of mono and gemini cationic surfactants was illustrated in Fig. . The monAEAA  was dissolved in dichloromethane. The organic solution was mixed with 14.31 g of 135 mmol of Na2CO3 aqueous solution. The resulting two-phase mixture was cooled to 5 °C. A solution of chloroacetyl chloride  in dichloromethane was added dropwise to the cooled solution for around 40 min while maintaining at 5 °C temperature. Then, the mixture was stirred for two hours at room temperature. The washing of the aqueous solution was done with dichloromethane three times, all of the organic solutions were added, then rinsed with water and dried over anhydrous Na2SO4 to get a brown waxy product43.In a 500 ml round bottom flask, N-(2-aminoethyl) alkyl amide 44.In a 250 ml round bottom flask, N--2-chloroacetamide (AECA) , and a solution of triethylamine  in ethyl acetate was added and stirring under reflux 24 h. Under reduced pressure, the solvent was eliminated, and the crude product underwent three washes with diethyl ether to produce a white precipitate; m.p: 91–96 °C. The product was dried and stored in desiccators to prevent moisture absorption45.The mixture of N,N,N′,N′-Tetramethylethane-1,2-diamine and N--2-chloroacetamide (AECA) in a molar ratio of 1:2 in ethyl acetate was refluxed for 48 h. A white precipitate was formed during the reaction and increased after completed the reaction time. The resulting product was washed with n-hexane and recrystallized in absolute ethanol to get an off-white precipitate with m.p. 98–102 °C. The product was dried and stored in desiccators to prevent moisture absorption1H & 13C-NMR at 400, and 100 MHz respectively: . Finally, the mass spectra were attained through a GC Ms-QP 1000 EX mass spectrometer  at 70 eV.The chemical structures of all synthesized compounds were confirmed by IR spectra  and were recorded on the CARY 630 FT-IR spectrometer . Also, NMR spectra were measured in (DMSO-d6) on a Bruker Avance (III) NMR spectrometer at 400 MHz; 46.The antimicrobial potential of mono and gemini cationic surfactants has been assessed according to the agar plate diffusion method47:The formulations of cutting fluid emulsions or emulsifiable soluble oil blends were prepared as followsA cutting oil package has been formulated from different ratios of vegetable base oil (recycled vegetable oil) (78–89 by wt.%), individually synthesized emulsifiers  (8–15 by wt.%) or mixed synthesized emulsifiers  (8–12 by wt.%), oiliness or lubricant (oleic acid) (1–4 by wt.%), anticorrosion and biocide (GCS) (1–3 by wt.%) and coupling agent (diethylene glycol) (1–3 by wt.%). The mixture was blended with each other by magnetic stirring. Each ingredient in this mixture was tested individually at various concentrations to find the optimal concentration at which oil blend stability occurs. The performance of prepared cutting oil formulas was assessed based on cutting oil package stability and emulsion stability.In accordance with the standard procedure (IP311), put the test oil package into specimen bottles. All of the bottles were sealed, and placed in the oven at 50 °C as well as 0 °C for the duration of at least 15 h and at most 20 h. After this period, remove the bottles and immediately inspect the oil for any indications of turbidity, separation, or gelling; the blend that produces stable oil  was chosen for further examination.In accordance with the standard procedure (IP 263), all blend samples were examined for emulsion stability in the following way: Before testing, bring the oil package and water to a temperature of 25 °C. Utilizing the magnetic stirrer, stir the required amount of water in a 100 ml conical flask at a speed enough to produce a vortex just deep enough to reach the flask's bottom. Add the required amount of oil quickly and stir continuously for 2 min. Pour a sufficient amount of the emulsion into the graduated-neck flask. This flask was left for 24 h to assess emulsion stability . Only emulsion-stable metal-cutting fluids (0.1 ml of cream or oil layer separation after 24 h) were chosen for further testing.In accordance with the standard procedure (ASTM D4627) for evaluating the anticorrosion test of metal cutting fluid emulsions, the cast iron chips were cleaned with benzene prior to use. Weigh 4.0 ± 0.1 g of cast iron chips and place them in a petri dish on filter paper. Put 5.0 ml of emulsions on cast iron chips. Use the stirring rod to ensure that all chips are submerged, all air bubbles have been released, and the chips are distributed evenly. The dish is covered and left to stand for 24 h. The amount of rust stain on the filter paper indicates the fluid's ability to control corrosion.The surface tension was assessed using the pendant drop technique utilizing Theta optical tensiometer, Biolin Scientific Company, Finland. The surface tension of various formulas was measured at 25 °C (298 K). To ensure the measurement's accuracy, water was examined.The sessile drop method was used to measure the contact angle with a Theta Optical Tensiometer, Biolin Scientific Company, Finland. The computer automatically fits the droplet profile and computes the contact angle. Polished carbon steel (CS), aluminum (Al) 6061, and tungsten carbide (WC) surfaces were used to mimic the carbon steel, aluminum-based workpiece, and the tool material respectively, and the contact angle was measured on each. Between tests, the sample surfaces were cleaned with ethanol and dried.The droplet size distribution of the prepared formulas was measured by using dynamic light scattering  at 25 °C.A ZEISS Axiolab 5 digital laboratory optical polarizing microscope equipped with a Leica MC190 HD microscope camera was utilized in order to perform photographic microscopy tests for FI, FVI and commercial sample at 25 °C (298 K). On a glass slide, an emulsion droplet was spread out and exanimated.A tribology measuring cell connected to Physica MCR-502 controlled-stress rheometers was used to conduct tribological tests. A ball-on-pyramid principle governs the setting of the tribology accessory A 6.35 mm diameter steel ball (1.4401 grade 100) rotating on three steel plates (1.4301) at a 45° angle is used in the cell. The evolution of the friction factor with sliding velocities was studied for the normal load (10 N) and 900 s at 25 °C.2) was maintained to allow the MTT to be metabolized. The media were then dumped off and MTT metabolic product was re-suspend formazan in 200ul Dimethyl sulfoxide (DMSO). Plates were placed on a shaking table (150 rpm for 5 min) to thoroughly mix the formazan into the solvent. Optical Density (O.D.) at 560 nm was recorded and subtracts background at 620 nm. The maximum non-toxic concentration [MNTC] of each extract was determined and was used for further biological studies48.In vitro, cytotoxicity of two MCF samples  was assessed by MTT assay. Samples were investigated for their cytotoxicity effect against human keratinocyte line (HaCaT cell line). For each sample, different concentrations ug/ml  were prepared. Growth medium was decanted from 96 well micro titer plates after confluent sheet of Vero-e6 cell was formed; cell monolayer was washed twice with wash media. Double-fold dilutions of tested sample were made using Dulbecco’s Modified Eagle’s Medium (DMEM), where 0.1 ml of each dilution was tested in different wells leaving 3 wells as control, receiving only maintenance medium. Microplates were incubated at 37 °C and examined for 48 h. Cells were checked for any physical signs of toxicity. MTT solution was prepared (5 mg/ml in PBS) (BIO BASIC CANADA INC), and then 20ul MTT solution were added to each well, and shaked at 150 rpm for five minutes, to thoroughly mix the MTT into the media. Incubation for 4 h .Recycled vegetable oil from SBE using n-hexane was 11%, which was 17–19% lower than the amounts reported in the other studies52. The oil was yellow in color, clear, and free of any suspended particles. While RO had a density comparable to refined soybean oil, its viscosity was 1.66 times higher, owing mostly to its increased amount of saturated fatty acids. The RO exhibited a saponification value (SV) of 209.30, surpassing that of the refined oil (198.95 mg of KOH/g of oil). Additionally, the RO had a significantly higher free fatty acid (FFA) content of 53.67%, in stark contrast to the refined soybean oil or recycled vegetable oil obtained from SBE as reported in the earlier studies49. The refined oil had an iodine value of 122.6 g/100 g, indicating a rather high degree of unsaturation3. In comparison to refined soybean oil, RO had the lowest iodine value of 64.7 g/100 g, owing to its higher percentage of saturated fatty acids. Additionally, the RO had a high peroxide value, indicating the existence of considerable amounts of hydro-peroxides generated via oil oxidation during the bleaching and storage operations53. It is also worth noting that RO oxidizes faster than refined oil, which can be related to FFAs' higher oxidation sensitivity relative to triglycerides54. Finally, the RO had a greater flash point, which contributed to a lower risk of smoke and fire dangers, hence improving safety in our specific application55.Table 3.The RO contained a notable proportion of saturated fatty acids, specifically palmitic acid (34.79%) and stearic acid (15.69%). In contrast, only 15% of the fatty acids in refined soybean oil were saturated. On the other hand, the RO exhibited significant amounts of unsaturated fatty acids, such as oleic acid (41.45%) and linoleic acid (8.08%), while refined soybean oil primarily consisted of unsaturated fatty acids (84.89%). The RO displayed a lower content of unsaturated fatty acids, particularly linoleic and linolenic acids, compared to refined soybean oil. This disparity can be attributed to the harsh conditions to which the SBE recycled vegetable oil was exposed, including light, oxygen, and high temperature, which accelerated the oxidation reactions of unsaturated fatty acids38. Subsequently, the HEAA was sulfated with chlorosulfonic acid, to obtain Sodium 2-alkylamidoethyl sulfate (AS)40. The chemical reaction was illustrated in Fig. The synthesis of an anionic emulsifier can be achieved in two steps. Firstly, recovered oil was subjected to amidation with monoethanolamine, using anhydrous sodium acetate as a catalyst to form N-(2-hydroxyethyl)alkyl amide (HEAA)νmax cm−1) , 3296.19 (νNH stretch), 2919.07, 2850.18(νC–H asym. and sym. stretch), 1642.49 .FT-IR  Fig. : 3472.991H-NMR spectrum : of HEAA ; 1.2 7); 1.40 ; 1.98 ; 2.13 ; 2.69  3.32 ; 3.57 ; 4.40 ; 5.26  6.9 .EAA Fig.  demonstrνmax cm−1) , 2922.00, 2851.85(νC–H asym. and sym. stretch), 1639.32 , 1549.37 (νC-N stretch), 1241.23, 1068.02, 1025.37 (νS=O asym. and sym. stretch.).FT-IR  Fig. : 3318.501H-NMR spectrum : of AS ; 1.3 7); 1.5 ; 1.97 ; 2.21 ; 2.77 ; 3.41 ; disappearance of proton of OH and increase chemical shift of CH2 at 4.09 ; 5.37 ; 7.52 .41. In the last step, nonionic emulsifiers (NS1-4) are obtained by reacting AEOB with polyethylene glycol of various molecular weights  in the presence of p-toluenesulfonic acid (PTSA) as a catalyst42. The chemical reaction was illustrated in Fig. The synthesis of nonionic emulsifiers can be accomplished in three steps. Firstly, the recovered oil was subjected to amidation with ethylenediamine using anhydrous sodium acetate as a catalyst to form N-(2-aminoethyl)alkyl amide (AEAA). Secondly, the reaction between (AEAA) and succinic anhydride produces 4-amino)-4-oxobutanoic acid (AEOB)νmax cm−1) , 2921.04, 2850.97(νC–H asym. and sym. stretch), 1640.87 .FT-IR  Fig. : 3304.241H-NMR spectrum : of AEAA ; 1.22 7); 1.3 ; 1.57 ; 2.03 ; 2.14 ; 2.84 ; 3.32 ; 4.04 ; 5.30 ; 6.98 .EAA Fig.  demonstr13C-NMR: spectrum of AEAA ; 22.66 (CH3CH2(CH2)); 25.61 ; 27.18 ((CH2)4CH2CH2CO, CH = CHCH2CH = CHCH2CH2); 29.63 ; 31.89 (CH2C = O); 36.63 (NHCH2CH2NH2); 40.01 (NHCH2CH2NH2); 128.28 (CH = CHCH2CH = CH); 130.20 (CH = CHCH2CH = CH); 174.66 (C = O).The EAA Fig.  illustra20H38N2O) with a base peak at 55.13 , and various fragments of this compound give the peaks with different intensities at different m/z positions as at 321.79 , 305.45 (44% C19H35N2O), 71.39 (30% C5H11), 43.86 (50% C2H6N), 43.14 (71% CHNO). The mass spectrum analysis affirmed the chemical composition of the synthesized AEAA.Mass spectrum Fig.  of AEAA νmax cm−1) , 3302.75 (νNH stretch), 2922.95, 2851.21 (νC–H asym. and sym. stretch), 1712.89 , 1642.23 , 1555.69 (νC-N stretch).FT-IR  Fig. : 3416.411H-NMR spectrum : of AEOB ; 1.27 9); 1.47 ; 2.03 6CH2C = O); 2.29 ; 2.43 ; 2.51 ; 2.74 ; 3.06 ; 5.32  ); 7.75 ; 7.84 .EOB Fig.  revealedνmax cm−1) , 2906.89, 2872.18 (νC–H asym. and sym. stretch), 1249.53, 1731.29 , 1649.18 , 1547.61 (νC-N stretch), 1105.62 (νC−O−C) ethereal band, asym. stretch).FT-IR  Fig. : 3423.941H-NMR spectrum : of NS ; 1.12 7); 1.41 4CH2CH2); 1.88 3CH2CH = CH, CH = CHCH2(CH2)4); 1.98 4CH2CH2); 2.43 ; 2.54 ; 3.28 ; 3.34 ; 3.45 ); 3.56 ); 3.67 , 3.68 ; 4.10 ; 4.80 ; 7.03 6CONH); 7.57 . NS Fig.  revealed2CO3 at 5℃. Then, chloroacetyl chloride is added to the cooled mixture and stirred at room temperature for 2 h, resulting in the formation of N--2-chloroacetamide (AECA)43. Subsequently, AECA reacts with a solution of triethylamine and N,N,N′,N′-Tetramethylethane-1,2-diamine in ethyl acetate, leading to the formation of mono and gemini cationic surfactants, respectively45. The chemical reaction was illustrated in Fig. (The synthesis of mono and gemini cationic surfactants as follows. N-(2-aminoethyl)alkyl amide (AEAA) is dissolved in dichloromethane and mixed with aqueous solution of Na in Fig. .νmax cm−1) , 2922.51, 2852.38 (νC–H asym. and sym. stretch), 1638.02 , 1555.87 (νC-N stretch).FT-IR  Fig. : 3284.761H-NMR spectrum : of AECA ; 1.30 7); 1.58 ; 2.01 ; 2.18 ; 2.76 ; 3.40 ; 4.00 ; 5.36 ; 6.59 ; 7.53 ; 8.14 .ECA Fig.  demonstrνmax cm−1) , 2921.04, 2851.38(νC–H asym. and sym. stretch), 1640.69 , 1555.56 (νC-N stretch).FT-IR  Fig. : 3305.731H-NMR spectrum : of MCS ; 1.31 9), ((CH3CH2)3N+); 1.62 6CH2C = O); 2.06 ; 2.20 3); 2.78 ; 3.40 ; 5.39 ; 6.52 .MCS Fig.  revealedνmax cm−1) , 2924.37, 2852.84 (νC–H asym. and sym. stretch), 1641.48 , 1548.91 (νC-N stretch).FT-IR  Fig. : 3276.881H-NMR spectrum : of GCS ; 1.22 9); 1.46 ; 2.04 6CH2C = O); 2.56 ; 2.71 2NCH2); 3.16 ; 3.31 2N+CH2); 4.23 2); 5.33 : 8.00 ; 8.97 2).GCS Fig.  revealed57.Metal cutting fluids may become contaminated by microorganisms from a variety of sources, including contaminated raw materials, improper cleaning techniques, airborne contamination, human contact, mixing with water, and system leaks. To limit microbial growth, biocides must be added to metal cutting fluid60.Cationic surfactants have good resistance towards microbial activity due to their positive charge which has a strong affinity for negatively charged the microbial cell membrane. Furthermore, the presence of these surfactants' long hydrophobic alkyl chains can interact with the hydrophobic lipid membrane. In addition, the counter anion may penetrate the cell's cytoplasm. These effects cause the cell's selective permeability to be disrupted, resulting in cell deathBacillus pumilis, Streptococcus faecalis, Escherichia coli, Enterobacter cloacae, Saccharomyces cerevisiae, and Candida albicans compared to Penicillin G, Ciprofloxacin, and Ketoconazole-respectively. The antimicrobial activity of the surfactants studied was first evaluated using the disc diffusion method. All of the microorganisms studied were inhibited by the solutions of each surfactant applied to the plates. All of the surfactants tested had inhibition zones ranging from 10 to 20 mm for all types of bacteria and fungi species, which were termed active61. The GCS and MCS displayed the highest anti-bacterial behavior  to E.coli and B. pumilis relative to the control  respectively. Whereas, the GCS and MCS displayed the highest anti-fungal behavior  to C. albicans compared to control (24 mm) respectively. According to the results, the tested compounds have significant anti-bacterial and anti-fungal activities towards all tested microorganisms, with the GCS having better anti-bacterial activity. Gemini cationic surfactants differ from mono-cationic surfactants in their molecular structure, where they have two positively charged groups instead of one. Because of this dual charge, gemini surfactants adsorb more strongly to negatively charged bacterial cell walls, disrupting the cell membrane and causing cell death60.In vitro results of antibacterial and antifungal activity of synthesized mono and gemini cationic surfactants are reported in Table  for variB. pumilis, S. faecalis, E. coli, and E. Cloacae using the micro broth dilution method. Remarkably, GCS had the lowest MIC and MBC values against E.coli  as shown in Table Gemini outperformed mono-cationic surfactants in terms of broad antibacterial activity against both Gram-positive and Gram-negative organisms. Therefore, the antimicrobial activity of the gemini cationic compound was quantitatively evaluated by measuring the minimum inhibitory concentrations (MICs), and the minimum bacterial concentration (MBC), for 35. This cutting oil package has been diluted with water to create an oil-in-water emulsion. The resulting fluid is milky white in color. The distinctive advantage of this kind of fluid is that it combines the cooling properties of water and the lubricating properties of oil63. Vegetable oil's highly lubricating properties can be attributed to the fundamental chemical composition of vegetable oil molecules (it contains a polar group). Where the polar group draws the vegetable oil molecule to a metallic surface creating a thick, strong, and durable lubricant film that reduces friction between the cutting tool and the workpiece during machining operations64.Emulsifiable soluble oils are formulated by blending vegetable base oil with emulsifying agents , lubricant oil (oiliness), biocide, corrosion inhibitor, and coupling agent65. All cutting fluid emulsion formulations include a corrosion inhibitor to prevent rusting and corrosion on metal surfaces. They act by creating a layer of protection on the metal's surface. The corrosion inhibitor can help to increase cutting efficiency, decrease maintenance costs, and lengthen the life of cutting tools21. Biocide is applied in cutting fluid emulsion formulations to inhibit the growth of microorganisms, which can lead to a variety of issues, including corrosion, odor, health risks, and decreased performance. Biocides are added in low concentrations to ensure their effectiveness and to minimize any potential negative effects on the cutting fluid itself66. Additionally, oiliness plays a role in providing better lubrication and improving the workpiece surface finish67. Finally, coupling agents are incorporated into every formulation to improve the compatibility and solubility of additives in the oil, resulting in enhanced performance and stability of the fluid63.The emulsifying agent plays a crucial role in stabilizing the cutting fluid emulsion by reducing the interfacial tension between the oil and water, forming a protective layer around each oil droplet, and reducing the droplet size, which prevents coalescence and separation of the oil from the waterTo achieve optimum cutting oil package stability, a series of cutting oil formulas with different ingredient ratios were created. A formula that gives a stable cutting oil package of homogeneous clear oil (without separation or indications of turbidity), as shown in Fig. Emulsifiable soluble oils are typically made by dispersing a cutting oil package in tap water in a volume ratio of 5/95. After 24 h of preparation, the stability of these emulsions was observed. The concentration of the incorporated components in all formulations has an impact on the metal cutting emulsion's stability and performance. It is clear from Fig. 68. Mixed emulsifiers exhibit diverse structures and properties (Mixed emulsifiers can have a variety of different structures and properties), influencing their capacity to form stable emulsions. For instance, one type of emulsifier may exhibit rapid adsorption and effective reduction of interfacial tension, but may not be as proficient at preventing droplet coalescence. Conversely, another emulsifier may not be as efficient in reducing interfacial tension but is highly efficient in inhibiting droplet coalescence. By combining emulsifiers with varying properties, it becomes possible to optimize the balance of interfacial forces and create a more stable emulsion. Consequently, mixed emulsifiers may have synergistic effects on emulsion stability68. Furthermore, the combination of a water-soluble emulsifier and an oil-soluble emulsifier in all cutting fluid formulations can have a synergistic effect on emulsion stability in terms of coalescence rates69. Therefore, the HLB of the mixed emulsifiers was calculated. The hydrophilic-lipophilic balance (HLB) value of a surfactant is typically used to express the surfactant's capacity to create an emulsion. Also, it plays a crucial role in emulsion stability71.By utilizing a blend of two or more diverse emulsifiers, rather than individual emulsifiers, the formation, stability, and performance of oil-in-water emulsions can be significantly improved72:AB, HLBA, and HLBB are HLB of mixed emulsifiers and individual emulsifiers respectively, A% and B% are the mass percentages of A and B in the mixed emulsifiers, respectively.For mixed surfactants, an effective HLB can be calculated using mass percentages and HLB numbers of the individual emulsifiers. The mixed HLB values were calculated by the following equation73.If the hydrophilic part of the emulsifier has only ethylene oxide, the HLB can be calculated for individual emulsifiers using the following formulaTo determine the optimum HLB value that gives a stable oil-in-water emulsion, a mixture of oil-soluble nonionic and water-soluble nonionic with different hydrophilic-lipophilic balance (HLB) values of 12, 11, 10, 9, and 8 was prepared. It is worth noting that the HLB value of 10 provided the best stability for O/W emulsions for nonionic/nonionic.A number of cutting oil package formulations with different constituent ratios have been prepared. Figure The cutting fluid emulsion was tested for stability over a period of 24 h. Typically, cutting oil packages are dissolved in tap water at a volume ratio of 5/95 to produce emulsifiable soluble oils. The stability and effectiveness of the metal-cutting emulsion are influenced by the concentration of the incorporated ingredients in all formulations. From the results illustrated in Table A corrosion test was performed for all stable soluble oil emulsions . Figure 74. The contact angle is a key parameter used to quantify wettability. The contact angle of a cutting fluid on a metal surface can significantly impact its effectiveness during metal cutting. A low contact angle indicates good wetting and spreading of the cutting fluid on the metal surface. This allows the fluid to form a continuous and uniform film, providing efficient cooling and lubrication75. The Young equation can be stated as follows:θ is the contact angle.Wettability plays a crucial role in metal-cutting fluids and the behavior of liquids on solid substrates. Wettability refers to the ability of a liquid to spread or adhere to a solid surfaceγLG) were found to be similar (31 and 29 for Formula I and Formula VI). Therefore, the disparity in contact angles observed between fluids is likely attributable to their varying interfacial tensions (γSL) with solid surfaces, as the (γSG) value remained constant in Eq.  on the workpieces (CS and Al), and tool (WC) are shown in Fig. 77.The droplet size of the dispersed phase in a cutting fluid nanoemulsion is an important parameter that can affect the performance of the fluid. In general, smaller droplets lead to improved cooling efficiency and lubrication, as they increase the surface area of the dispersed phase and enhance the contact between the fluid and the workpiece. Additionally, smaller droplets can reduce the tendency of the fluid to clog the cutting tool and can improve the overall stability of the fluid78. FVI displayed smaller droplet sizes and higher droplet size uniformity than the FI. This is attributed to the negatively charged head group of anionic emulsifier in FVI which creates electrostatic repulsion impeding the close approach and coalescence of the droplet. Several studies have shown that the use of emulsifier mixtures can lead to a reduction in droplet size and an improvement in emulsion stability compared to using a single surfactant78.The mean droplet size indices for the prepared emulsions, formulas I and VI, were measured at 25 °C using the DLS method for the nano-emulsions produced with different mixtures of emulsion-stabilizing surfactants. As shown in Fig. 71. Formula VI produces better outcomes in surface tension, wettability, droplet size, and photographic image testing. As a result, Formula VI was chosen to go through additional testing.According to the photographic images in Fig. 79. Material and energy conservation is becoming an essential problem. Friction is the fundamental reason for energy loss in a mechanical system, but it can be reduced with lubrication. As a result, it is critical to improve the lubrication properties. The key to optimizing this procedure is a good blend of oil base and additives81.A change in metal-cutting technology has resulted from recent tribological developments over the past few years82. As the film's thickness and adhesion increase, the friction coefficient depresses83.The friction coefficients for the commercial sample, FVI are displayed in Fig. 18 evaluated the performance of two formulations, a green cutting fluid and commercial sample on HaCaT cell lines. No death was recorded on using the green at 50 ug/ml, whereas the commercial sample caused a significant cellular mortality at 50 ug/ml18.HaCaT cell lines were tailored as a model system to examine the potential toxicity of cutting fluids  in human keratinocyte cell lines. For FVI, MTT results showed its maximum toxicity value 32.396251673%) at 10,000 ug/ml followed by 15.662650602% at 5000 ug/ml. On the other hand, no toxicity was recorded for FVI at 2500, 1250, 625, and 312.5 ug/ml after 48 h. Furthermore, the used commercial sample caused 37.148594378% and 18.005354752% at 10,000, and 5000 ug/ml respectively, and no toxicity was recorded at 2500, 1250, 625, and 312.5 ug/ml. Recorded IC50 values were 13,520.76 and 12,570.30 ug/ml for FVI and commercial sample respectively as shown in Table 1673% at The recycled vegetable oil was extracted from SBE via the cold method and its physiochemical properties were assessed.The recycled vegetable oil was modified chemically to synthesize anionic and nonionic emulsifiers with various ethylene oxide units, as well as mono and gemini cationic surfactants as corrosion inhibitors and biocides.According to the results, mixed emulsifiers with different ratios of nonionic/nonionic , and anionic/nonionic  displayed stable emulsions when compared to individual emulsifiers.Formulas I and VI showed good protective efficiency from the corrosion test.The prepared eco-friendly formula VI possessed better wettability  and a smaller particle size (63.97 nm).Additionally, the impact of formula VI on the machine's performance was examined. According to the findings, formula VI was able to display that the friction coefficient decreases with increasing sliding velocity.The cytotoxicity assay demonstrated the safety of formula VI on humans and the environment.Supplementary Figures.


Comparing the long-term tumor control results of partial cystectomy(PC)and radical cystectomy(RC)in the treatment of muscle-invasive bladder cancer, and to explore the feasible method of bladder preservation therapy (BPT)in patients with MIBC.We retrospectively analyzed the clinical data of 102 patients with muscle-invasive bladder cancer in our hospital between January 2012 and December 2018, of whom 32 cases in the partial cystectomy group and 70 cases in the radical cystectomy group. We performed a comparative analysis of patient general information, perioperative-related indicators and postoperative follow-up data, comparing OS, PFS, and DSS at 1, 2, 3, 4, and 5 years in both groups, and comparing tumour recurrence and metastasis in postoperative patients.All the 102 cases in this study were successfully completed. Partial cystectomy group and Radical cystectomy group median operating time (169.50(130.00 ~ 225.25) min and 420.00(343.75 ~ 483.75) min, p < 0.001), median intraoperative blood loss was (100(50 ~ 100)ml and 400(200 ~ 1000)ml, p < 0.001), median perioperative blood transfusion volume (0(0 ~ 0)ml and 600(150.00 ~ 906.25)ml, p < 0.001), median total hospital stay (18(14.25 ~ 20.00) and 24.5(20.00 ~ 34.25) days, p < 0.001), median preoperative preparation time (7(4.25 ~ 8.00) and 10(8.00 ~ 13.00) days, p < 0.001), median postoperative hospital stay (9(8.00 ~ 13.50) and 14(11.00 ~ 21.25) days, p < 0.001), the incidence of perioperative blood transfusion was , the incidence of surgical complications was(28.1%(9/32) and 50.0%(35/70), p = 0.033), average hospitalization cost ((26435.76 ± 9877.82) yuan and (58464.36 ± 19753.13) yuan, p < 0.001), the differences were statistically significant (p < 0.05). Perioperative mortality (0 vs. 2.9%(2/70), p = 1), and OS at 1, 2, 3, 4, and 5 years after surgery were , PFS . DSS , and the incidence of tumor recurrence or metastasis was (40.0%(12/30) vs. 25.4%(16/63), p = 0.151), the differences were not statistically significant (p > 0.05).In patients with limited solitary T2N0M0 and T3N0M0 muscle-invasive bladder cancer, partial cystectomy plus bladder instillations treatment can achieve comparable tumour control to radical cystectomy. However, patients in the PC group have significant advantages in terms of operative time, intraoperative bleeding, intraoperative and postoperative blood transfusion, preoperative preparation time, total hospital stay, postoperative recovery time, operative costs and operative complications. This option may be considered for such patients with a need for bladder preservation. Bladder cancer is a common malignant tumor in the urinary and male reproductive system, seriously endangering human health, about 30% of the first diagnosis is muscle-invasive bladder cancer (MIBC) . RadicalCurrent guidelines support the use of triple (TURBT + radiotherapy + chemotherapy) therapy as an alternative to RC, but emphasise the need for appropriate patient selection, close multidisciplinary collaboration between surgeons, radiologists and oncologists, and long-term monitoring, so the chances of recurrence remain high , 8. TripPartial cystectomy as a surgical procedure that both preserves bladder function and completely removes the lesion. It has been widely used in the treatment of MIBC since the mid-20th century. However, due to the lack of strict patient screening and different surgeon levels, the survival rate of patients at 5 years after surgery was low and the postoperative recurrence rate was high . As RC hWe retrospectively collected patients diagnosed with muscle-invasive bladder cancer in the Urology Department of Lanzhou University Second Hospital between January 2012 and December 2018. This study received the Lanzhou University Second Hospital Ethical Committee approval(2017 A-053). Patient inclusion criteria were: (1) initial diagnosis of patients with muscle-invasive bladder cancer; (2) patients with pathological stage T2-T3 without lymph node metastases and distant metastases; (3) Patients with muscle-invasive bladder cancer with localized solitary origin and less than 2 lesions. (4) postoperative pathology suggestive of uroepithelial carcinoma;(5)patients with complete case information. Exclusion criteria: (1) patients with recurrent muscle-invasive bladder cancer; (2) tumours near the neck of the bladder; (3) patients with small volume bladders; (4) patients who have had radiotherapy to the lower abdomen; (5)Patients with muscle-invasive bladder cancer with high risk of recurrence after comprehensive evaluation.The patients in this group underwent PC (open and laparoscopic surgery) and immediate and maintenance bladder irrigation with epirubicin or hydroxycamptothecin after surgery. Open surgery is performed through a median or curved incision in the abdomen. A 2 cm skin incision is made below the umbilicus, a 10 mm Trocar is placed, the pneumoperitoneum is connected and the pressure is controlled at 12–15 mmHg, the laparoscope is placed, a 10 mm and a 5 mm Trocar are placed on both sides of the external border of the rectus abdominis muscle under direct vision and the corresponding laparoscopic instruments are placed. The tumour is removed together with 2 cm of surrounding normal tissue. If the resection includes the uretero-vesical junction on one side, a simultaneous uretero-vesical reimplantation with a double J-tube should be performed. Intraoperative surgery, the internal pelvic lymph nodes, including the internal iliac, external iliac lymph nodes, and surrounding the obturator nerve, were explored, combined with preoperative imaging examination, and the suspicious enlarged lymph nodes were cleaned and sent for pathological examination.In this group, conventional bladder instillations therapy was administered 4 weeks after surgery with pirorubicin or hydroxycamptothecin. Post-operative bladder instillations is 1 time per week for 8 times and then 1 time per month for 12 times. Pirorubicin was 30 mg for every time and hydroxycamptothecin was 20 mg for every time.2 injection, abdominal pneumoperitoneum pressure to 12 ~ 15mmHg, puncture 10 mmTrocar, puncture 10 mmTrocar in the left and right inferior umbilical rectus ventral muscle respectively, puncture 5 mmTrocar in the right and left anterior superior iliac spine 2 ~ 3 cm above and medial respectively, and the corresponding laparoscopic surgical instruments were inserted. Radical resection of the bladder and surrounding adipose tissue, distal ureter, men including the bladder, prostate, seminal vesicle, women underwent anterior pelvic visceral enucleation, including the bladder, uterus, fallopian tubes, ovaries. Part of the urethra and the anterior wall of the vagina are removed when infiltration into the bladder triangle or posterior urethra , Average hospitalization costs was , The median intraoperative blood loss was measured between , The median perioperative blood transfusion volume was , The median total length of stay was , The median postoperative length of stay was measured between , The median preoperative preparation time was , The incidence of perioperative blood transfusions was , the differences were all statistically significant (p < 0.05). There was no significant difference in the perioperative mortality rates . Perioperative period data is shown in Table 25.25min In the PC group, four postoperative patients underwent hydroxycamptothecin bladder instillations therapy, the remaining 28 patients were given pirorubicin bladder instillations chemotherapy.There were 7 21.9%), 2 (6.1%) and 1 (3%) cases of intravascular tumour embolism or invasion, nerve invasion and histological variation respectively in the pathological specimens of the PC group; there were no cases of multiple tumours, 29 (90.7%) in the T2N0M0 stage and 3 (9.3%) in the T3N0M0 stage. Among the pathological specimens in the RC group, there were 28 (40.0%), 11 (15.7%) and 7 (10.0%) of intravascular tumour embolism or invasion, nerve invasion and histological variation respectively; there were no cases of multiple tumours, 65 (92.9%) had stage T2N0M0 and 5 (7.1%) had stage T3N0M0. There were no significant differences in TNM stage, intravascular tumour embolism or invasion, nerve invasion, and histological variation (p > 0.05). See Table .9%, 2  months, and the RC group with a median follow-up of 22.5 (12.25 to 45.25) months. OS at 1, 2, 3, 4 and 5 years after surgery in the PC and RC groups were ; PFS were ; DSS was , respectively, and the incidence of tumour recurrence or metastasis were , respectively, and none of the differences were statistically significant (p > 0.05)  of the PC group had recurrence or metastasis. There were 6 patients with distant metastases, including 1 small bowel metastasis who died more than 2 months after reduction surgery, 1 lung metastasis, 1 pharyngeal metastasis and 3 lymph node metastases, for which no special treatment was given. Six patients had local recurrences, two of which had muscular infiltration and were treated with salvage RC, one of whom died of multiple metastases more than 6 months after surgery, and one of whom was treated with postoperative gemcitabine plus cisplatin (GC) chemotherapy and survived despite urethral metastases, as the tumour was controlled by timely detection and complete excision. Two patients were treated with TURBT after postoperative detection of recurrence in the bladder, one died shortly after recurrence, one eventually developed pancreaticobiliary cancer and died, and the other two patients died after recurrence without special management. As the study was retrospective, there was not complete uniformity in terms of treatment strategy, while some patients and families refused radical bladder cancer surgery and failed to achieve timely salvage RC treatment for patients with recurrence in the bladder detected early as recommended by the guidelines , which mIn this study, we found that patients with partial bladder resection were not statistically significant in tumor recurrence or distant metastasis and bladder cancer-specific death, which may not have a longer follow-up time, with some limitations, and a longer follow-up time is needed to clarify the clinical efficacy of partial bladder resection. In addition, considering that this study is a retrospective single-centre study, the small sample size affects the reliability of the evidence and a multi-centre, large sample size, randomised controlled study could be conducted subsequently to verify the feasibility of this treatment option.In patients with limited solitary T2N0M0 and T3N0M0 muscle-invasive bladder cancer, partial cystectomy plus bladder instillations treatment can achieve comparable tumour control to radical cystectomy. In our study, patients in the PC group have significant advantages in terms of operative time, intraoperative bleeding, intraoperative and postoperative blood transfusion, preoperative preparation time, total hospital stay, postoperative recovery time, operative costs and operative complications. This option may be considered for such patients with a need for bladder preservation.