Influenza prevention remains a significant public health concern that is still not adequately addressed by vaccines or current therapeutic options. Cidara has developed CD388, a multivalent conjugate of a dimeric NAI with a proprietary hIgG1 Fc domain engineered for extended half-life. CD388 is in clinical development (NCT05285137 and NCT05523089) for the prevention of seasonal influenza A and B. Herein we analyze the contribution of Fc-mediated immune effector function to CD388 efficacy.-/- mice. The Fcer1g-/- mice are deficient in activating FcγRs thereby excluding contribution of Fc-mediated immunity to efficacy. After lethal challenge with influenza A virus, CD388 or analogues were administered subcutaneously or intramuscularly two hours post-infection. Animals were monitored daily for 14 days or longer for survival (< 20% BW loss). For viral burden quantification, lungs were harvested on day 4 post-infection and viral titers were determined by plaque assay.CD388, Fc engineered to extend PK, and closely related Fc modified analogues (immune-active ‘IA’ or immune-silent ‘IS’) were generated. CD388-IA can engage Fc gamma receptors (FcγRs) whereas IS fails to engage FcγRs required to trigger Fc-mediated immune effector functions (e.g. antibody-dependent cellular cytotoxicity). Efficacy studies were conducted in BALB/c WT or Fcer1g-/- mice at the same doses required for protection in WT mice. However, a CD388-IA analogue at low DAR of 1, that has reduced antiviral activity, demonstrated improved efficacy in WT mice as compared to KO mice .CD388-IA and -IS at comparable drug to antibody ratios (DARs) of 4.5±0.5  demonstrated comparable protection  and comparable dose-dependent viral burden and cytokine reduction in a lethal mouse model (Table 1). Additionally, the CD388-IA analogue at DAR 4.7 was protective in Fcer1gThese data combined demonstrate that the efficacy of CD388 at DAR 4.5±0.5 is driven predominantly by the intrinsic antiviral activity of the small molecule NAI and is independent of the contribution of Fc-mediated effector functions.Simon Döhrmann, PhD, Cidara Therapeutics: Stocks/Bonds James Levin, PhD, Cidara Therapeutics: Stocks/Bonds Elizabeth Abelovski, B.S., Cidara Therapeutics: Ownership Interest Amanda Almaguer, Bachelors, Cidara Therapeutics: Stocks/Bonds Rajvir Grewal, n/a, Cidara Therapeutics: Ownership Interest Karin Amundson, BSc, Cidara Therapeutics: Stocks/Bonds Joanne Fortier, BSc, Cidara Therapeutics: Stocks/Bonds Thanh Lam, PhD, Cidara Therapeutics: Stocks/Bonds Thomas P. Brady, Ph.D., Cidara Therapeutics: Stocks/Bonds Allen Borchardt, PhD, Cidara Therapeutics: Stocks/Bonds Jason N. Cole, Ph.D., Cidara Therapeutics: Stocks/Bonds Leslie W. Tari, Ph.D., Cidara Therapeutics: Stocks/Bonds


Furthermore, the activation energy of the proton conduction of the P–MDP composite was approximately 0.18 eV. These results suggested that the proton conduction of the P–MDP composite was based on an anhydrous proton conductive mechanism. In contrast, the anhydrous proton conduction of the P–methanediphosphonic acid (MP) composite, which did not form the self-assembled lamellar structure, was ca. 3 × 10−5 S cm−1 at 120–130 °C and this value was one order of magnitude lower than that of the P–MDP composite. Therefore, the two-dimensional self-assembled proton conductive pathway of the P–MDP composite plays a role in the anhydrous proton conduction.We prepared a protamine–monododecyl phosphate composite by mixing protamine (P) and a monododecyl phosphate (MDP). This P–MDP composite formed an acid–base complex by the electrostatic interaction between cationic protamine and the negatively charged phosphate group. Additionally, according to the X-ray diffraction (XRD) measurements, the composite formed a self-assembled lamellar structure with an interaction between the long alkyl chains of MDP. As a result, the P–MDP composite showed the proton conductivity of 9.5 × 10 We prepared an anhydrous proton conductive protamine–monododecyl phosphate composite by mixing protamine (P) and a monododecyl phosphate (MDP). Therefore, electrolyte membranes consisting of a fluorine-free polymer, such as a hydrocarbon polymer or biopolymer, that exhibit the proton conduction at an intermediate temperature under anhydrous or low humidity conditions, have been reported.6–10The proton conductor has received attention as an electrolyte membrane of the polymer electrolyte fuel cell (PEFC) without the emission of carbon dioxide. Especially, the PEFC, which is operated at intermediate temperatures (100–200 °C) or under anhydrous conditions, has many advantages, such as a higher energy efficiency, improved CO tolerance of the Pt electrode, fuel management, and co-generation.11 The protamine, one of the proteins, is obtained from fish milt, which is discarded as an industrial waste around the world, and is a strong basic biopolymer containing a large amount of the basic amino acid arginine (Arg).12,13 Therefore, a material consisting of protamine can be produced at low cost and its strong basic property can be generated at the proton defect sites in the material. Additionally, since the protamine is a biopolymer, a material consisting of protamine is not only environmentally benign but also biodegradable, making it easy to dispose of after use. Thus, we prepared a composite consisting of the strong basic protein protamine and phosphonic acid and reported its utilization as a proton conductor. As a result, the protamine and methanediphosphonic acid (MP) formed an acid–base complex through the electrostatic interaction between the guanidino group in Arg and the phosphate group in MP and had the proton conductivity of 3 × 10−4 S cm−1 at 120 °C under anhydrous conditions. This composite material consisting of protamine can be expected to exhibit a higher anhydrous proton conductivity by improving the preparation method of the composite.Recently, we prepared a protamine-acidic molecule composite material and reported its anhydrous proton conduction.14–20 These proton conducting pathways are formed by the phase separation of polymers, the surface of nano fiber, the crystalline ion channels, or the self-assembly of surfactant molecules with long alkyl chains.14–20 In this case, since the distance between neighboring functional groups is extremely short, the proton transfer easily occurs along the proton conducting pathway. In addition, the proton conductive mechanism in the pathway is a Grotthuss mechanism in which protons transfer from the proton donor site to proton accept site, rather than a vehicular mechanism in which protons move with diffusible vehicle molecules, such as the H3O+ or H5O2+.21,22 As a result, materials with the proton conducting pathway exhibit a high proton conduction under anhydrous or low humidity conditions.14–20 Therefore, in this research, we prepared the protamine composite with a two-dimensional proton conducting pathway using self-assembled methods and evaluated its anhydrous proton conduction. The protamine composite with the two-dimensional proton conducting pathway will exhibit a higher anhydrous proton conductivity than the P–MP composite without the self-assembled structure.On the other hand, proton conductors with the proton conducting pathway in the electrolyte membrane have been reported.−4 S cm−1 at 120–130 °C under anhydrous conditions. This proton conductivity was more than one order of magnitude higher than that of the P–methanediphosphonic acid (MP) impregnated glass filter which did not form the self-assembled structure.In this study, the protamine-monododecyl phosphate (P–MDP) composite was prepared by mixing the protamine (P) and monododecyl phosphate (MDP). These materials formed an acid–base complex through the electrostatic interaction between the guanidino group in Arg and the phosphate group in MDP. According to X-ray diffraction (XRD) measurements, the P–MDP composite formed the self-assembled lamellar structure. Additionally, the phosphate groups in the self-assembled structure behave as a proton conducting pathway. As a result, the P–MDP impregnated glass filter showed the proton conductivity of 9.5 × 102.2.1.Protamine sulfate , monododecyl phosphate (MDP), and methanediphosphonic acid (MP) were purchased from Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan or Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan. These reagents ware used without the purification. 2.2.11The purchased protamine sulfate possesses the sulfate ion as a counter ion in its protein. These inorganic ions in the composite can be an obstacle to proton conduction measurements. Therefore, the sulfate ion was exchanged with the hydroxide ion by an ion exchange column using the strongly basic ion exchange resin, Amberlite® IRA-400. The exchange to hydroxide ions was confirmed by a pH measurement. The ion-exchanged aqueous protamine solution was freeze-dried for more than 48 hours and used for subsequent experiments.−1) and the P–MDP mixed solution was heated at 70 °C for 5 minutes. This P–MDP mixed solution was cast on a polytetrafluoroethylene (PTFE) plate and dried overnight at room temperature. These P–MDP composites were used as samples for an infrared (IR), a thermogravimetric-differential thermal analysis (TG-DTA), and an X-ray diffraction (XRD) measurements. In contrast, the samples for the proton conduction measurement were prepared by impregnating a glass filter with the P–MDP composite. This preparation was as follows: the P–MDP mixed solutions were cast on a glass filter (7 × 7 mm2) and dried overnight at room temperature.The P–MDP composite was prepared as follows: the protamine and MDP were dissolved in ethanol/water  and ethanol, respectively. In a screw-capped microtube, the MDP solution was added to a protamine solution (50 mg mLR) of the MDP in the P–MDP composite was defined by R value was 0–1. On the other hand, the P–MP composites were also prepared by a similar procedure.The molar ratio . The measurement was done at the heating rate of 10 °C min−1 under flowing dry-nitrogen. The sample weight was normalized at 1 mg. The X-ray diffraction (XRD) patterns of the composites were measured by a SmartLab  or a Philips X'pert  at a power level of 45 kV and 200 mA with Cu-Kα radiation (λ = 1.54 Å) in the 2θ/θ scanning mode.The molecular structure of the P–MDP composite was determined by the attenuated total reflection (ATR) methods using an FT/IR-4700 infrared spectrophotometer  with the diamond ATR prism. The IR spectrum was measured at the resolution of 4 cm2.4.20 The electrode samples were placed in a stainless steel vessel, heated at 130 °C for 2 hours under flowing dry-nitrogen to evaporate the water and volatile components, then cooled to room temperature under flowing dry-nitrogen. The proton conductivity of the P–MDP composite was measured by the a.c. impedance method using a 3532-80 chemical impedance analyzer  from room temperature to 130 °C under flowing dry-nitrogen. The frequency range in the proton conductive measurement was from 4 Hz to 1 MHz.11,20 The resistance and proton conductivity of the P–MDP composite was determined from analysis of the Cole–Cole plots.The proton conduction of the P–MDP composite was measured as follows: the P–MDP composite impregnated glass filter was sandwiched with two gold electrodes (5 mm diameter).3.3.1.R = 0.5 composite, (c) R = 0.7 composite, (d) R = 0.9 composite, and (e) MDP without the composite. The protamine without the composite had an absorption band at 1620 cm−1, related to the stretching vibration of N–H.11,23–25 This absorption band decreased by the addition of MDP, and at the same time, a new absorption band appeared at 1637 cm−1. Since the absorption band at 1637 cm−1 was attributed to the stretching vibration of the –NH3+ group,11,23 the amino groups present in the guanidino group of protamine were protonated by the addition of MDP. In contrast, the MDP without the composite showed an absorption band at 1024 cm−1, related to the asymmetric stretching vibration of the P–OH group.11,23,26,27 This absorption band relatively decreased with the addition of protamine. Additionally, a new absorption band at 953 cm−1 and 1054 cm−1, attributed to the deprotonation of the P–OH group,11,23,26,27 appeared by the addition of protamine. Consequently, the addition of protamine to the MDP induced the deprotonation of the P–OH group in MDP. These results suggested that the P–OH group in the MDP molecules and the amino site of the guanidino group in the protamine behave as the acidic and basic molecules, respectively, and the protamine and MDP form the acid–base complex through the electrostatic interactions between the P–O− group and the –NH3+ group. Similar results, such as the complex formation by an electrostatic interaction, have been reported for various anhydrous acid–base proton conductors.11,20,28The molecular structure of the P–MDP composite was evaluated using an IR spectrometer with the diamond ATR prism. 3.2.−1 under flowing dry-nitrogen. The sample weights were normalized at 1 mg. R = 0.7 composite, (3) R = 0.9 composite, and (4) MDP without the composite, respectively. All samples showed an endothermic peak at <100 °C. These endothermic peaks were due to the evaporation of water contents and appeared as the TG weight loss at <100 °C. Additionally, in the protamine without the composite, the TG curve showed a further TG weight loss above 200 °C and the DTA curve showed an exothermic peak at approximately 200 °C. These results suggested that the thermal decomposition of protamine without the composite occurred above 200 °C. In contrast, the MDP without the composite exhibited a large endothermic peak and a high TG weight loss at around 200 °C. This is due to the dehydrated reaction of the phosphate group and similar results have been reported.11,20,29 This endothermic peak related to the dehydrated reaction was shifted to a higher temperature by the addition of protamine. The reason for these phenomena is as follows: since the guanidino groups in protamine and the phosphate groups in MDP form an acid–base complex through an electrostatic interaction, the addition of protamine increases the distance between the non-deprotonated P–OH groups in MDP. As a result, a higher temperature was required for the dehydration between the distant P–OH groups. Additionally, at high R values, such as 0.9 ≤ R, an exothermic peak appeared at around 185 °C. This phenomenon can be postulated of as follows: the protons of MDP added in excess behave as acidic catalysts in the composite. As a result, the thermal decomposition of protamine occurred at a lower temperature. In fact, at R = 0.95, the exothermic peak appeared at an even lower temperature  R = 0.7, (◯) R = 0.8, (△) R = 0.9, (■) R = 0.93, (●) R = 0.95, and (▲) R = 0.98 composites under flowing dry-nitrogen  R = 0.7, (◯) R = 0.8, (△) R = 0.9, (■) R = 0.93, (●) R = 0.95, and (▲) R = 0.98 composites are shown in Ea) based on the proton conduction was calculated from the slope of the straight line.11,20,30 These Ea values are shown in Ea values decreased with the increased R values and showed the minimum value at R = 0.95, which indicated the maximum proton conductivity. Generally, the Ea value of the proton conductor based on the vehicular mechanism is ca. 0.02 eV.21,31,32 The Ea values obtained in our experiment were almost one order of magnitude higher than this value. Additionally, the P–MDP composite possesses no mobile ions other than protons in its composite. These results suggested that the proton conducting mechanism of the P–MDP composite was a Grotthuss-type mechanism with the intermolecular proton transfer and the P–MDP composites acted as an anhydrous proton conductor.The Arrhenius plots of the proton conduction of (◇) 3.4.R = 0.95 composite, and (c) protamine without the composite in the low angle region. The insert shows the XRD patterns in the high angle region. The protamine without the composite did not show any diffraction pattern. Thus far, it has been reported that the MDP molecule formed a lamellar structure.33,34 In our research, the MDP without the composite indicated the diffraction patterns at 3.2°, 6.5°, and 9.9°. These diffraction peaks are related to the lamellar structure which were assigned to (100), (200), and (300). These distances are 27.4 Å, 13.5 Å, and 8.94 Å, respectively, and these distances almost coincided with the reported distances.33,34 Additionally, the diffraction pattern at 23.7° appeared as the distance between the alkyl groups and this distance is 3.9 Å. These results suggested that the MDP without the composite formed a lamellar structure with the d-spacing of ca. 27 Å and the distance between the phosphate groups was ca. 4 Å. Similar results were also obtained for the R = 0.95 composite. In this case, the R = 0.95 composite formed a lamellar structure with the d-spacing of ca. 26 Å and the distance between the alkyl groups was ca. 4.6 Å. For the R = 0.95 composite, the values of the d-spacing and the distance between the alkyl groups were slightly different from that of MDP without the composite. This is due to the distortion of lamellar structure by the addition of protamine. These results suggested that the MDP with the addition of protamine retained a lamellar structure. Since the phosphate groups in the R = 0.95 composite are regularly arranged and the distance between the phosphate groups is ca 4.6 Å, these phosphate groups might function as a two-dimensional proton conducting pathway.3.5.R = 0.33, all the protons of MDP were deprotonated and all the guanidino groups were protonated. Therefore, P–MDP showed no proton conduction at low R values, such as R ≤ 0.5. At high R values, such as 0.6 ≤ R, although all the guanidino groups form the protonated guanidino groups, the phosphate groups exist as a mixture of deprotonated and non-deprotonated phosphate groups. These aggregates of phosphate groups play a role in the proton conductor under anhydrous conditions.Since the MDP can donate two protons and the guanidino group can accept one proton, at d-spacing of ca. 26 Å. In addition, the distance between the alkyl chains of MDP was ca. 4.6 Å. + in R values, such as 0.6 ≤ R, the MDP molecules exist as a mixture of deprotonated phosphate groups, such as P–O−, and non-deprotonated phosphate groups, such as P–OH. In addition, since the MDP forms a self-assembled structure in the composites and the distance between the phosphate groups is extremely short, the MDP behaves as a two-dimensional self-assembled proton conducting pathway  composite, which cannot form a self-assembled structure, was 3 × 10−5 S cm−1 and this value was more than one order of magnitude lower. By using a protamine, which is discarded as an industrial waste around the world, it is possible to create proton conducting materials at a lower cost than artificial polymers. In addition, the protein, such as protamine, is not only an environmentally benign and biodegradable materials but also a sustainable material. Therefore, the anhydrous proton conductive protein–MDP composite with the lamellar structure has a potential to use for electrodevices, such as biofuel cells and biosensors, in the biomedical, bioengineering, and environmental fields. Furthermore, since the protein composite materials are highly safe for human, they might be applied for the actuators that can be used within the human body.The P–MDP composite was prepared by mixing the protamine (P) and monododecyl phosphate (MDP). These P–MDP composites formed the acid–base composite by electrostatic interactions between the negatively charged phosphate group and positively charged guanidino group. In addition, according to the XRD, the P–MDP composite formed a self-assembled structure along the long alkyl chain. Since the distance between the phosphate groups was extremely short, these MDP molecules acted as a two-dimensional proton conducting pathway. As a result, the P–MDP composite of Masanori Yamada: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, writing – review & editing. Naoaki Yoshihara: formal analysis, software.There are no conflicts to declare.RA-013-D3RA07191J-s001


Functional nucleic acid (FNA) probes have been widely used in environmental monitoring, food analysis, clinical diagnosis, and biological imaging because of their easy synthesis, functional modification, flexible design, and stable properties. However, most FNA probes are designed based on one-photon (OP) in the ultraviolet or visible regions, and the effectiveness of these OP-based FNA probes may be hindered by certain factors, such as their potential for photodamage and limited light tissue penetration. Two-photon (TP) is characterized by the nonlinear absorption of two relatively low-energy photons of near-infrared (NIR) light with the resulting emission of high-energy ultraviolet or visible light. TP-based FNA probes have excellent properties, including lower tissue self-absorption and autofluorescence, reduced photodamage and photobleaching, and higher spatial resolution, making them more advantageous than the conventional OP-based FNA probes in biomedical sensing. In this review, we summarize the recent advances of TP-excited and -activated FNA probes and detail their applications in biomolecular detection. In addition, we also share our views on the highlights and limitations of TP-based FNA probes. The ultimate goal is to provide design approaches for the development of high-performance TP-based FNA probes, thereby promoting their biological applications. Nucleic acids have gained prominence as a fundamental framework of functionally diverse and versatile molecular probes due to their unique molecular recognition capabilities, programmable base sequences, convenient synthesis and modification, and stimuli-triggered responsiveness ,2,3,4. FBased on the many advantages of FNA molecules, extensive efforts have been made in the development of FNA probes used in detecting a diverse array of analytes , such asTypically, most FNA probes were primarily designed based on one-photon (OP) that is required to be excited/activated by a short wavelength light source in the ultraviolet or visible (UV/Vis) regions ,42. OP-bIn comparison with OP-based FNA probes that employ a short wavelength light source, FNA probes based on two-photon (TP) can be excited/activated by a long wavelength femtosecond laser ,49,50. TThis review  providesFNA probes based on one-photon excitation (OPE) suffer from limitations such as limited tissue penetration depth, photodamage, and photobleaching. To overcome these challenges, two-photon excitation (TPE) fluorescence imaging has become an optimal approach for enhancing sensing capabilities in living cells and tissues. In contrast to the prevailing construction strategy of OPE-based FNA probes, TPE-based FNA probes constructed by introducing TP dye as the signal reporter can effectively eliminate the problems related to shallow imaging depth and background fluorescence interference in biological matrices while using an NIR excitation wavelength that provides increased penetration depth and reduced photodamage. Therefore, the applications of TPE-based FNA probes are extensive in the field of biosensing, allowing for the detection of biomolecules such as nucleic acids, enzymes, biothiols, ATP, and metal ions. By investigating the dynamics and interactions between molecules, the underlying mechanisms of pathophysiological processes can be better attained, thereby advancing disease diagnosis and therapeutic monitoring.MicroRNAs (miRNAs) are short noncoding nucleic acid fragments, typically comprising 21–23 nucleotides, that play crucial roles in gene expression regulation and cellular functions . MiRNAs In general, TPE-based FNA probes were designed by incorporating TP dye molecules that could be excited by a TP laser to produce fluorescence signals. As shown in To accurately measure the length of DNA molecules, Yuan et al. conducted a study where they prepared assemblies of DNA-tuned AuNPs with controlled separation distances to investigate the impact of plasmon coupling strength and particle size on two-photon photoluminescence (TPPL) enhancement c 69]. T. T69]. TEnzymes are specific and efficient biocatalysts, most of which are proteins produced by living cells that perform essential roles in living systems by accelerating biochemical reactions through their highly efficient catalytic activities ,71,72. DAccording to the different hydrolysis characteristics of enzymes, different nucleic acid sequences that responded to the hydrolysis of enzymes were designed on the FNA probes. As shown in Biothiols, which encompass thiol-containing amino acids and peptides, serve as vital structural or functional constituents in many proteins and peptides and fulfill various essential functions in biological systems ,84. GSH,As shown in Similarly, Liu et al. fabricated AgNPs/DNA-TP dye conjugates as a TPE-based nanoprobe for imaging biothiols in living cells b 92]. T. T92]. TAdenosine 5′-triphosphate (ATP), an essential cellular component present in all living organisms, serves as an indispensable intracellular energy source and plays a role as a signaling molecule associated with energy status ,94,95. IAs shown in Metal ions are vital components in biological systems, where they play critical roles in numerous biological processes ,107,108.2+ by utilizing an RNA-cleaving DNAzyme coupled with TP fluorophores . . 129]. TAs with the previously mentioned RNA assays, traditional ATP detection methods also face the issue of requiring a large amount of sample, which masks the heterogeneity between cells . HoweverAs shown in Typically, TP-based FNA probes are composed of FNA sequences along with fluorophores or linkers that are sensitive to TP irradiation. The FNA sequences serve a crucial role in specific interactions with target biomolecules and can include aptamers, DNAzymes, modified nucleic acids, and other related elements. TP technology serves a dual purpose in TP-based FNA probes. Not only does it function as an excitation light source for TP dye, producing fluorescence signals, but it can also serve as an activation light source, enabling spatiotemporal control over FNA probes. As a result, TP-based FNA probes offer advantages such as high spatiotemporal resolution, deep tissue penetration, low photodamage, and high sensitivity. These probes have garnered significant attention in the field of studying molecular interactions in cells, monitoring and imaging drug delivery processes, and quantifying biomarkers. In addition, they are also expected to be used in the regulation of biomimetic materials, the synthesis of artificial tissues, and the preparation of biological hydrogels.Despite the years of development and utilization of TP-based FNA probes in biomedicine, they are still considered to be in their early stages. For the further development of high-performance TP-based FNA probes and the promotion of their commercialization, there are still some limitations with TP-based FNA probes that should be addressed. Priority should be given to the development of TP dye molecules with higher TP excitation efficiency and environmental stability, thereby improving the imaging resolution and reliability. Additionally, there is a need to develop a user-friendly TP platform that can achieve different regulation effects by adjusting the wavelength, power, and spot size of the two-photon laser. Lastly, efforts should focus on developing additional FNA probe vectors to improve their delivery and targeting efficiency.


Arabidopsis thaliana) anthers, the tapetum monolayer undergoes cell differentiation to support pollen development. Here, we report that a transcription factor, DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1), inhibits tapetal cell division leading to cell differentiation. We identified SKEWED5-SIMILAR 18 (SKS18) as a downstream target of TDF1. Enzymatic assays showed that SKS18, annotated as a multicopper oxidase-like protein, has ascorbate oxidase activity, leading to AsA oxidation. We also show that VITAMIN C DEFECTIVE1 (VTC1), an AsA biosynthetic enzyme, is negatively controlled by TDF1 to maintain proper AsA contents. Consistently, either knockout of SKS18 or VTC1 overexpression raised AsA concentrations, resulting in extra tapetal cells, while SKS18 overexpression in tdf1 or the vtc1-3 tdf1 double mutant mitigated their defective tapetum. We observed that high AsA concentrations caused lower accumulation of reactive oxygen species (ROS) in tapetal cells. Overexpression of ROS scavenging genes in tapetum restored excess cell divisions. Thus, our findings demonstrate that TDF1-regulated AsA balances cell division and cell differentiation in the tapetum through governing ROS homeostasis.The major antioxidant L-ascorbic acid (AsA) plays important roles in plant growth, development, and stress responses. However, the importance of AsA concentration and the regulation of AsA metabolism in plant reproduction remain unclear. In Arabidopsis ( AsA accumulation mediated by TDF1 controls the transition from cell division to differentiation in the tapetum through governing ROS homeostasis Background: The tapetum is the innermost layer of the 4 cell-layered anther wall. In Arabidopsis, the tapetum undergoes cell differentiation and transitions into secretory cells. Most nutrients for microspores and/or developing pollen grains are produced by, stored in, and transported from this secretory tapetum. Therefore, the tapetum of flowering plants plays a critical role in mature pollen production. DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) is a tapetum-specific transcriptional factor. In tdf1 mutants, tapetal cells fail to differentiate after the establishment of the anther wall, leading to complete male sterility.Question: How does TDF1 promote tapetum differentiation?Findings: We found that the loss of TDF1 function induces supernumerary cell divisions and disturbs the subsequent differentiation in the tapetum. We show that the TDF1-SKS18 module regulates the L-ascorbic acid (AsA) oxidation pathway and that the TDF1-VTC1 module regulates AsA biosynthesis. Genetic analyses supported the notion that TDF1 inhibits extra tapetal cell divisions via mediating proper AsA contents. Further studies revealed that AsA-mediated reactive oxygen species (ROS) homeostasis modulates cell divisions in the tapetum. Thus, our findings reveal that TDF1-regulated AsA concentrations contribute to the transition from division to differentiation of tapetal cells through governing ROS homeostasis.Next steps: We will further investigate other pathways or mechanisms that function in tapetal cell division and/or differentiation. Moreover, we will explore whether other SKS members are required for the regulation of tapetum development.2O2) via the activity of plant-specific ascorbate peroxidase (APX) in the symplasm , a highly abundant metabolite, has many important roles in growth and metabolism for both plants and animals. In humans, AsA is indispensable for health, since deficiency in AsA causes scurvy . In plansymplasm . Of notesymplasm , AsA is symplasm .Arabidopsis thaliana), a vtc1-1 mutant harboring a point mutation in the gene only contains 25% to 30% of wild-type (WT) AsA levels, supporting its critical role in AsA biosynthesis (Zea mays) roots proposed that AAO oxidizes AsA to maintain quiescent center (QC) cells in a state of reduced mitotic activity . The tapetum, the innermost layer of a 4-layered anther wall, acts as a nursing tissue that provides nutrients and materials to microspores and/or developing pollen grains . In Arabtdf1 mutant have a swollen shape with additional vacuolization and fail to transition to secretory tissue after the anther wall was established  genes cooperatively form a tapetal regulatory pathway (DYSFUNCTIONAL TAPETUM1 [DYT1]-TDF1- ABORTED MICROSPORES [AMS]- MALE STERILE188 [MS188]-MALE STERILITY1[MS1]) to control tapetum development and function . Among tablished . HoweverSKU5 SIMILAR 18 (SKS18) encoding a multicopper oxidase (MCO)-like protein functioning in AsA oxidation with a copper cofactor. Additionally, TDF1 regulates AsA biosynthesis via negatively controlling the accumulation of VTC1. Our physiological, biochemical, and genetic evidence demonstrated that TDF1 prevents the supernumerary division of tapetal cells and enables tapetal differentiation via mediating a proper AsA level. We further showed that a high level of AsA leads to low ROS levels in the tapetum. Consistently, increasing the expression of genes encoding ROS scavenging enzymes restored excess tapetal cell divisions seen in WT anthers. Conversely, a low level of AsA allowed ROS to accumulate and repress tapetal cell divisions. Together, our findings reveal that TDF1-regulated AsA concentration contributes to the transition from division to differentiation of tapetal cells through governing ROS homeostasis.Here, we show that TDF1 is a negative regulator of AsA accumulation in anthers. TDF1 directly activates the expression of tdf1 mutant is defective in early tapetum development in Arabidopsis (tdf1 at Stages 6 to 9 by transmission electron microscopy (TEM). At Stage 6, the WT tapetal cells were binucleate and cytoplasmically condensed  of the SKS gene family are categorized into the group of miscellaneous enzymes; this group is the most enriched group in the functional classification of these candidates  was a direct target of TDF1  assay using inflorescences from tdf1 gTDF1pro:TDF1-GFP transgenic plants. TDF1 was reported to bind to the sequence AACC(T/A/C) -TDF1 in Escherichia coli. We determined that MBP-TDF1 can indeed bind to the biotin-labeled probe. Importantly, unlabeled competitor probes or labeled GGGG-containing mutated probes incubated with MBP-TDF1 abolished this shift, confirming the specificity of the binding (SKS18 promoter.To identify whether TDF1 directly controlled C(T/A/C) . We scan samples . Quantit samples . We also binding . These rSKS18 activation, we carried out transient dual-luciferase (LUC) assays in Arabidopsis protoplasts. Since TDF1 and SKS18 were co-expressed in anthers (SKS18pro:LUC) and effector (35S:TDF1) into protoplasts. The empty vector was included as a control. Co-infiltration of 35S:TDF1 and SKS18pro:LUC in Nicotiana benthamiana leaves resulted in strong LUC intensity, demonstrating that TDF1 activates SKS18 transcription  /CRISPR-associated nuclease 9 (Cas9)-mediated gene editing  . We conf RT-qPCR ; this mu RT-qPCR . We perf Stage 6 . From St Stage 6 , whereas transgenic lines in the T1 generation. We chose several independent lines to measure their SKS18 expression levels by RT-qPCR: SKS18 transcript levels in these transgenic plants were at least 6 times higher than in tdf1 plants  to stain fatty acids in the tapetum. Like in WT, we observed a fluorescent signal in the tapetum from tdf1 SKS18-OE #4 anthers  product (Cucurbita spp.) callus markedly increased by adding copper  transgenic lines generated, 2 independent lines (VTC1-OE #4 and VTC1-OE #5) with high VTC1 expression showed compromised pollen viability  staining  transgenic lines. Among these lines, APX1-OE #21 had a higher expression of APX1 than the WT, showing severe pollen abortion (DYT1550pro:CAT3-GFP (named CAT3-OE) lines exhibited compromised male sterility with the higher expression of CAT3  and CATALASE 3 (CAT3) are 2 important Hn plants . To addrabortion . Similar of CAT3 . We dete of CAT3 . CytologE plants , suggestSKS18 in WT plants. Among these DYT1550pro:SKS18-GFP transgenic lines (named SKS18-OE), SKS18-OE #30 had the highest expression level of SKS18  TF, is essential for restricting cell divisions after tapetal cells are specified . The ortivisions . The dat tapetum . The ini tapetum . One nuc tapetum . Howeverokinesis , randomlokinesis , and M. okinesis , which iis  Cys2/His2-type zinc-finger protein, directly binds to CSN5B to allow the accumulation of VTC1  Columbia-0 (Col-0) and Landsberg erecta (Ler) accessions were used in this study. The homozygous tdf1 (ams (sks18-1 (WiscDsLoxHs032_08H) and vtc1-3 (SAIL_611_D10.v1). The double mutant, vtc1-3 tdf1, was generated by a genetic cross. Primer sequences for genotyping are listed in −2 s−1 (Philips Lifemax Cool White fluorescent bulbs) under long-day conditions (16-h light/8-h dark) in a growth room at 24 °C. For the measurement of AsA levels, vtc1-3 was grown under short-day conditions as described . For semi-thin sections, inflorescences from different genotypes were fixed for 2 to 3 d in FAA  and were embedded in Spurr's resin. Semi-thin sections (1 μm in thickness) were stained in a 0.01% (w/v) toluidine blue/sodium borate solution at 45 °C for 5 min. After washing off the excess solution with water, the sections were photographed with an Olympus BX51 microscope in a bright field.Individual anthers were dissected and stained with Alexander's solution in darkness for 2 h at room temperature . The statdf1 inflorescences were placed in ice-cold 2.5% (v/v) glutaraldehyde in 10 mM phosphate buffer (pH 7.4). Samples were postfixed in 1% (w/v) osmium tetroxide and dehydrated in an ethanol/water series . Samples were dehydrated twice with 100% propylene oxide and subsequently transferred to propylene oxide/Spurr's resin mixtures with ratios of 1:1, 1:3, and 3:1 (v/v), respectively. Later, samples were embedded in Spurr's resin and polymerized at 65 °C for 48 h. Ultrathin sections (50 to 70-nm thick) were stained in a solution of uranyl acetate and lead citrate. The images were viewed by a JEM-1230 TEM.For TEM, buds from WT and 2, 5 μL/mL) for 5 min as described  solubilized in 10% (v/v) glycerol for 2 to 3 h at 4 °C in the dark. Samples were subsequently mounted on slides and visualized with an Olympus FV3000 laser scanning microscope. For monitoring of FM4-64 signal, the 514-nm laser line was used for excitation and a 620- to 680-nm bandpass filter was used for detection. For analysis of tapetum development, semi-thin sections were orderly stained with toluidine blue (10 mg/mL) for 5 min, tinopal (10 μg/μL) for 15 min, and 3,3′-diethyloxacarbocyanine iodide  staining of H2O2 in anthers was carried out as described  staining of 2DCF-DA fluorescence was visualized by a filter set with 488 nm excitation and 498 to 532 nm bandpass. DHE fluorescence was visualized by a filter set with 518 nm excitation and 606 nm bandpass. The fluorescence intensities were quantified using ImageJ software. Based on the ROS fluorescent images, tapetal ROS intensities represent the mean value from all tapetal cells in 1 abaxial locule, calculated from about 10 independent images. Experiments were performed at least 3 times with different batches of plants. ROS fluorescent images used to produce these data are classified into 7 groups and provided in 2′,7′-dichlorodihydrofluorescein diacetate . The resulting amplicon was digested by restriction enzymes  and ligated into a modified GFP-pCAMBIA1300 vector . This construct was transformed into sks18 homozygous plants. To obtain the overexpressing lines, the genomic sequences of SKS18, VTC1, APX1, and CAT3 (without stop codons) were individually amplified and recombined into a modified GFP-pCAMBIA1300 vector, where their expression was driven by the DYT1 promoter (550 bp)  was amplified and cloned into the modified GFP-pCAMBIA1300 vector. This construct was transformed into Col-0 and tdf1/TDF1 plants, respectively. The genomic sequence of VTC1 without the stop codon plus its native promoter  was amplified and cloned into the modified GFP-pCAMBIA1300 vector. This construct was transformed into Col-0 and tdf1/TDF1 plants. The genomic sequence of TDF1 including the stop codon plus its native promoter (816 bp) was amplified and cloned into the modified VENUS-pCAMBIA1300 vector. This construct was transformed into Col-0 plants. All constructs were confirmed by DNA sequencing, introduced into Agrobacterium (Agrobacterium tumefaciens) strain GV3101, and transformed into plants by floral dipping  . These c dipping . All traSKS18 were inserted into the AtU6-26-sgRNA vector. The sgRNA cassette was digested with the enzymes NheI and SpeI, and ligated into the SpeI site of the ProYAO:hSpCas9 construct to generate the CRISPR/Cas9 vector. This construct was transformed into WT plants via Agrobacterium-mediated floral dipping. T1 transgenic plants were confirmed by PCR and were crossed with their respective backgrounds to obtain Cas9-free plants with mutations in the T2 progeny.CRISPR/Cas9 vectors were constructed as previously described . Two desSKS18 was measured by RT-PCR for 28 cycles. The qPCR reactions were carried out using a Real-time PCR System (Applied Biosystems) with a SYBR Green Real-time PCR Master Mix . At least 3 biological replicates and 3 technical replicates were performed for each combination of cDNA samples and primer pairs. β-TUBULIN served as an internal control. All PCR experiments were performed under the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 10 s, and 60 °C for 1 min. Under the same conditions, the ΔCt values were calculated and report the 2−ΔCt values as the fold-enrichment relative to β-TUBULIN. The primer sequences are listed in Root, stem, rosette leaves, inflorescences after plant bolting, and 21-d-old seedlings were harvested and quickly frozen in liquid nitrogen. Total RNA was extracted with a Trizol kit . First-strand cDNA synthesis was performed using Fly First-Strand cDNA Synthesis SuperMix  following the manufacturer's protocol. The transcript level of SKS18 cDNA fragment and a 449-bp VTC1 cDNA were amplified and cloned into the pBluescriptSK vector. These plasmids were individually digested by BamHI or EcoRI and used as templates. Sense and antisense probes were transcribed using the above templates by the T3 or T7 RNA polymerase , respectively. The hybridization for SKS18 transcripts in WT and tdf1 anthers was performed in a single batch, while that in ams was performed in another batch. The hybridization for VTC1 transcripts in WT and tdf1 anthers was performed in a single batch. Primer sequences are provided in Inflorescences were fixed in FAA buffer containing 3.7% (v/v) formaldehyde and vacuum-infiltrated for 15 min on ice. Samples were dehydrated in a graded ethanol series and stained with safranine in xylene/ethanol solutions. Samples were placed into a 60 °C oven for 1 wk and finally embedded in Paraplast . Transverse sections of 8 μm in thickness were transferred onto poly-L-lysine coated glass slides  for hybridization. RNA in situ hybridization was performed using a Digoxigenin RNA Labeling Kit . A 497-bp tdf1 gTDF1pro:TDF1-GFP complemented plants frozen in liquid nitrogen was crosslinked in 0.4 M sucrose-1% (v/v) formaldehyde buffer. Nuclei were isolated with extraction buffer and lysed with lysis buffer. The chromatin was sheared with sonication, resulting in most DNA fragments having a size between 200 and 800 bp. After pre-absorption using pre-immune serum with sheared salmon sperm DNA/protein A agarose mix  for 1 h, the DNA–protein complex was immunoprecipitated at 4 °C overnight using an anti-GFP monoclonal antibody  (1:100 dilution). Seventy microliters of magnetic beads coupled with protein G  were added to precipitate the antibody–protein/DNA complexes. After washing, the samples were incubated at 65 °C overnight to reverse the crosslinking. The co-precipitated DNA was purified and analyzed by qPCR as described above. Under the same conditions, the ΔCt values (Ct of each sample—Ct of the No antibody control) were calculated and reported 2−ΔCt as the fold enrichment. Primer sequences are listed in The ChIP procedure was performed as described with minor modifications. One gram of closed buds from TDF1 and SKS18 were cloned into the pMAL-p5X vector  and pCold TF DNA vector , respectively. These constructs were transformed into E. coli Rosetta Competent Cells  for protein production and purification. The transformed cells were cultured at 37 °C until the OD600 reached 0.6 to 0.8; protein production was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and growth at 18 °C overnight. Following centrifugation at 8,000×g for 5 min at 4 °C, cells were lysed by sonication on ice and supernatants were collected. For the MBP-TDF1 fusion protein, the supernatant was purified on Amylose resin . For the SKS18-His fusion protein, the supernatant was purified on Ni-NTA resin  according to the manufacturer's instructions. The purified proteins were quantified via SDS-PAGE and A280 absorbance using BSA as a standard, respectively. Primer sequences are listed in The coding sequences of AsA contents were measured using an ascorbic acid assay kit II . Freshly collected closed buds (anthers before Stage 12) (10 mg) were homogenized in cold Ferric Reducing/Antioxidant and Ascorbic Acid (FRASC) buffer. The homogenate was centrifuged at 13,000×g for 10 min at 4 °C and the supernatants were diluted 10 times in a final volume of 100 μL for AsA determination. To each sample, 10 μL of water was added, while 10 μL of ascorbic acid oxidase was added to the blank wells. The reaction was preincubated for 15 min at room temperature. A master reaction mix  was added to each sample. After incubation for 2 min, AsA content was determined by measuring the absorbance at 593 nm. The value from the blank sample (containing AAO) was subtracted from all readings to correct for background. AsA levels were then calculated based on an AsA standard curve. Similar results were obtained in at least 3 biological replicates.AsA contents were also measured using an Agilent 1290 Infinity II series UHPLC System (Agilent Technologies) equipped with Waters BEH C18 . Freshly collected closed buds were incubated with 500 μL extraction solution  and sonicated for 5 min in an ice-water bath 3 times. After centrifugation at 13,400×g for 15 min at 4 °C, the supernatants from each sample were extracted and diluted 100-fold with extraction solution. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was acetonitrile. The gradient of mobile phase B was as follows : 0 min, 5%; 2 min, 5%; 4 min, 90%; 6 min, 90%; 7 min, 5%; and 11 min, 5%. The flow rate was 300 μL/min. The column temperature was set to 35 °C and the auto-sampler temperature was set to 10 °C. The injection volume was 1 μL. An Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies), equipped with an AJS electrospray ionization (AJS-ESI) interface, was used for the assay. LC/MS-MS assays were performed by Lixinheng Technology Co., Ltd .2+, which shows a decrease in absorbance at 593 nm due to AsA oxidation in the reaction mixture containing Fe3+. The reaction system consisted of 0.06 mM AsA , 10 μM CuSO4, and the master reaction mix . The addition of recombinant purified SKS18-His protein (113 kD) to the sample wells initiated the reaction. At different time points, the reaction mixture was dispensed into a cuvette and its absorbance measured using a spectrophotometer (Eppendorf). Reactions with ascorbic acid oxidase  were used as a positive control. For negative controls, an equivalent volume of purified protein from E. coli cells harboring the empty pCold TF DNA vector was added.Ascorbic acid oxidase activity was determined by monitoring the formation of FeThe 5′ biotin-labeled probes used in this study were synthesized by Generay (Shanghai). EMSA was performed using a Lightshift Chemiluminescent EMSA Kit . Recombinant purified MBP-TDF1 protein was incubated with biotin-labeled probes in binding buffer . Competition experiments were performed by adding unlabeled DNA probes or labeled mutated probes. Each 20-μL binding reaction was incubated at room temperature for 20 min before separation on a 6% polyacrylamide gel in 0.5 × Tris-borate EDTA (TBE) buffer at 100 V for 90 min, after a pre-electrophoresis of 60 min. Separated samples were transferred onto a nylon membrane at 380 mA for 30 min. After crosslinking under UV light for 2 min, the subsequent experiments were conducted according to the manufacturer's protocol. The signals from the probes were captured with a Tanon-5500 Chemiluminescent Imaging System . Primer sequences are listed in SKS18  was PCR amplified from Col-0 and inserted into the pGreenII-0800-LUC vector. The resulting reporter construct contains 2 cassettes—the target promoter driving a firefly luciferase (LUC) reporter gene and a CaMV 35S promoter driving the Renilla luciferase (REN) gene as an internal control. To generate the effector constructs 35S:TDF1 and 35S:AMS, the TDF1 and AMS coding sequences were amplified and cloned into pCAMBIA1300-35S vector. Rosette leaves from Arabidopsis plants grown for 28 d were harvested and digested in an enzyme solution consisting of 0.8 M mannitol, 1 M KCl, 0.2 M MES (pH 5.7), 1.5% (w/v) cellulase R10, and 0.4% (w/v) macerozyme R10 . After sequencing, the above plasmids were transfected into mesophyll cell protoplasts via polyethylene glycol and incubated at room temperature for 16 h in the light. Following lysis, the supernatants were extracted from the transfected protoplasts. The firefly and Renilla luciferase activities were quantified using a Dual-Luciferase Assay Kit  and detected with a Synergy 2 multimode microplate (Bio-Tek) according to the manufacturer's instructions. The ratio of LUC/REN activities is shown. Primer sequences are listed in The promoter fragment of TDF1 (At3g28470), SKS18 (At1g75790), VTC1 (At2g39770), KEULE (At1g12360), DYT1 (At4g21330), AMS (At2g16910), APX1 (At1g07890), CAT3 (At1g20620).Sequence data from this article can be found in TAIR under the following accession numbers: The following materials are available in the online version of this article.. Extra tapetal cells in tdf1, tdf1 SKS18-OE #4, and sks18-1 anthers.. Localization of KEULE-GFP in KEULEpro:KEULE-GFP transgenic plants.. Expression analysis of SKS18 and identification of the sks18-2 mutant.. SKS18 transcript and SKS18 protein accumulation patterns in tdf1 and ams mutants.. TDF1 is a nucleus-localized protein in tapetal cells.. sks18-2 exhibits extra tapetal cells in anthers.. Overexpression of SKS18 partially rescues the defective tapetum seen in tdf1.. Pollen wall material biosynthesis in tdf1 SKS18-OE transgenic plants.. SDS-PAGE analysis of recombinant SKS18 protein.. AsA content measurements.. Extra tapetal cells observed in the VTC1-OE transgenic plants.. Expression analysis of VTC1 in the tdf1 mutant.. Analyses of tdf1 anthers.. SKS18-OE transgenic plants have fewer tapetal cells.. Multiple protein sequence alignment of SKS family proteins in Arabidopsis.. Primers used in this study.. Summary of statistical analyses.. ROS fluorescent images.. Multiple protein sequence of SKS family in FASTA format.koad037_Supplementary_DataClick here for additional data file.


Cancer cells frequently change their metabolism from aerobic glycolysis to lipid metabolism and amino acid metabolism to adapt to the malignant biological behaviours of infinite proliferation and distant metastasis. The significance of metabolic substances and patterns in tumour cell metastasis is becoming increasingly prominent. Tumour metastasis involves a series of significant steps such as the shedding of cancer cells from a primary tumour, resistance to apoptosis, and colonisation of metastatic sites. However, the role of glutamine in these processes remains unclear. This review summarises the key enzymes and transporters involved in glutamine metabolism that are related to the pathogenesis of malignant tumour metastasis. We also list the roles of glutamine in resisting oxidative stress and promoting immune escape. Finally, the significance of targeting glutamine metabolism in inhibiting tumour metastasis was proposed, research in this field improving our understanding of amino acid metabolism rewiring and simultaneously bringing about new and exciting therapeutic prospects. Behind Distant metastasis, a unique biological feature of malignant tumours, is associated with advanced tumour staging and poor prognosis . MetastaTargeting glutamine metabolism is beneficial for reducing tumour metastasis. This study focused on glutamine-metabolising enzymes and transporters to reveal the effect of glutamine on tumour cell metastasis . At the 2Since the 1950s, it has been noted that tumour cells consume large amounts of glutamine . A serie2.1Glutaminase (GLS), one of the most important enzymes in glutamine metabolism, is a component of glutamine utilisation that participates in the TCA cycle, producing ATP and NADPH, and maintains glutathione homeostasis and reactive oxygen species (ROS) in cells. Mammals mostly have two isozymes, GLS1 and GLS2. Although most cell lines co-express the transcripts of the two genes, the effects of the two types of GLSs on tumour metastasis are different.2.1.1in vitro and tumour growth and metastasis in vivo . Fi. Fi25]. 2.1.2Compared to GLS1, GLS2 is more diverse in promoting or inhibiting tumour metastasis. Zhang et al. found that GLS2 reduces the invasiveness of HCC cells in many ways. First, GLS2 can competitively bind to the small GTP enzyme Rac1 with guanine nucleotide exchange factor, an agonist of Rac1, and then inactivate Rac1, thus promoting P53-mediated tumour metastasis inhibition . Second,in situ showed more lung metastatic nodules when the tumour size in situ was the same; this is related to GLS2 promoting ERK/EGF/ZEB1 axis, which finally leads to an increase in EMT-related molecule-vimentin expression and promotes tumour metastasis [However, Ramirez-Peña et al. observed through the TCGA and ICGC databases that higher GLS2 expression in breast cancer indicated increased tumour metastasis. The experimental data verified this hypothesis. Compared with the control group, the experimental group injected with tumour cells that overexpressed GLS2 tastasis . In oesotastasis .2.2With the rise and deepening of glutamine metabolism research, SLC1A5 (ASCT2) has gradually become known as the main glutamine transporter, and increasing evidence has shown that SLC1A5 plays important roles in tumour metastasis. These include mediating ATP production and glutathione synthesis induced by glutamine, alleviating tumour cell growth under nutritional pressure, and endowing tumour cells with chemotherapy drug resistance . In a coin situ in the nude mice was significantly reduced, and lung and liver metastases were significantly reduced [In addition, Wang et al. reported that after the prostate cancer cells of shASCT2 were subcutaneously injected into nude mice, under the control of other variables, the tumour size  reduced . In addi reduced . In a st reduced . Zhuang  reduced . MucoepiRecently, a tumour suppressor gene, NDRG2, has been identified that belongs to the downstream gene family of N-Myc . Tumour 2.3SLC7A5, a glutamine reverse transporter, plays a role in the reverse transport of intracellular glutamine and extracellular leucine. In previous studies, SLC7A5 has been used as a marker for the independent prognosis of multiple tumours. High expression was observed in metastatic samples from more than ten cancers, including metastatic gastric cancer, endometrial-like cancer, advanced laryngeal squamous cell carcinoma, pulmonary neuroendocrine tumour, tongue cancer, colorectal cancer, cholangiocarcinoma, human epithelial ovarian cancer, non-small cell lung cancer, and human clear cell renal cell carcinoma . In addiTP53. In addition, miR-6775-3p can inhibit SLC7A5 expression.In gastric cancer, a new oncogene, GSE1, can positively regulate the expression of SLC7A5 in human gastric cancer cells by increasing the stability of the SLC7A5 transcript through a post-transcriptional regulatory mechanism and promoting tumour cell proliferation and metastasis, which was lost after knockdown by shSLC7A5 . In a stInterestingly, inhibition of miR-6775-3p and SLC7A5 can directly induce p53 transcription. The interaction between the two forms of positive feedback further inhibits the tumour and delays metastasis progression . Finally2.4in vitro and in vivo [Glutamate dehydrogenase (GDH), an important ring in the glutamine metabolic pathway, reversibly oxidises and deaminates glutamic acid to α-ketoglutaric acid (α-KG), which serves as the key catalytic enzyme for glutamine to enter the TCA cycle. While developing lung cancer-targeted drugs, Liu et al. found that GDH was upregulated by pleomorphic adenoma gene 1 (PLAG1). Its product α-KG, activated CaMKK2 by enhancing its indirect binding with AMPK. Simultaneously, CaMKK2 activated downstream AMPK, finally providing anti-anoikis and pro-metastatic signals in LKB1-deficient lung cancer, resulting in a poor prognostic outcome for LKB1-deficient lung cancer patient . Interes in vivo .Furthermore, it was found that compared with the simple expression of a certain genotype, high expression of GDH can also be combined with other factors to more effectively indicate poor prognostic outcomes, such as tumour metastasis. For example, there was no significant correlation between individual expression of GDH and SLC25A13, but higher expression of GDH in combination with lower expression of SLC25A13 was more strongly associated with tumour invasion than individual expression When glucose depletion . In a stin vitro GDH inhibitor R162 has also been used to overcome drug resistance and extensive in vivo metastasis. In addition to clarifying the mechanism, it also provides potential treatment strategies [In a study on drug resistance and EMT-mediated tumour metastasis in non-small cell lung cancer, Wang et al. selected GDH as a common contributing factor. It is believed that NSCLC increases oxidative phosphorylation by overexpressing GDH, leading to high resistance, and triggering migration and invasion by inducing Snail. The rategies . Howeverrategies .2.5Glutamate-oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) are the most important transaminases in the body. Their function has been fully elucidated in a series of tumours and non-tumours. As the most common biochemical indicators in clinical practice, they have multiple biological functions such as indicating liver function, releasing the degree of inflammatory factors, evaluating drug toxicity, and revealing tumour prognosis. However, their function is yet to be fully described and clarified in aspects such as tumour metastasis.In oral squamous cell carcinoma (OSCC), Li et al. compared extracellular vesicles derived from human primary tumour cells and matched lymph node metastatic (LN1) cells and a related database, and identified seven factors, including GOT1, in 670 kinds of proteins, 217 types of miRNAs, 26 kinds of metabolites, and 63 kinds of lipids, associated with decreased survival rate and increased tumour invasiveness in patients with cancer . In anotin situ and metastatic cancer cells and their death [However, in a study on the inhibition of cancer progression, researchers confirmed that α-ketoglutarate esters elicit death of OXPHOS-deficient cancer cells by elevating intracellular aKG and thereby sequestering nitrogen from aspartate through GOT1, leading to the interruption of oxidative phosphorylation progression in ir death . In addiir death . Interesir death ,67.2.5.1+ cells had a higher potential for liver metastasis. Simultaneously, they found that the cell line upregulated various metabolic enzymes, including GPT1. A relationship between GPT1 upregulation and metastasis to specific tumour sites was reported [In a study on the metastatic potential of lung cancer, researchers isolated a subpopulation of cells labeled with CD133 from A549 cell lines by flow cytometry and demonstrated that CD133reported . In anotreported .10.13039/100004915TCA cycle catch-up to support cell proliferation and metastasis during glutamine deprivation or GLS inhibition. Addition of the antioxidant N-acetylcysteine (NAC) significantly reduced the increase in GPT2 caused by glutamine deprivation or GLS inhibition. Finally, the combined effects of GLS1 and GPT2 in response to compensatory proliferation and distant metastasis of cells during nutritional crisis have been elaborated [Regarding the specific signalling pathway, control cells showed increased phosphorylation of the mTORC1 substrate p70s6K and hyperphosphorylated 4EBP1 protein enrichment compared to GPT2 knockout cells in a study involving triple-negative breast cancer. GPT2 knockdown can lead to impaired activity in tumour cell mTORC1, which promotes autophagy and reduces the proliferation and metastasis of tumour cells . In anotin vitro and promoted tumourigenesis and metastasis in mice in vivo. GPT2 inhibits proline hydroxylase 2 (PHD2) activity by regulating HIF1α stability and reducing α-KG levels. The GPT2-α-KG-PHD2 axis leads to the accumulation of HIF1α, constituting the sonic hedgehog signalling pathway. This eventually leads to tumour progression and metastasis [Many studies have focused on the interaction between hypoxia-inducible factor (HIF) and GPT2, which leads to tumour metastasis. In a study on HIF responding to hypoxia and promoting tumour cell metastasis by activating the transcription of glutamine metabolic enzyme in tumour cells, it was pointed out that in human glioblastoma (GBM), hypoxia could bind to the hypoxia response element of human GPT2 gene through hypoxia-inducible factor 1 (HIF1) rather than HIF2, leading to increased GPT2 transcription and promoting tumour cell proliferation and metastasis under hypoxia. Genetic or pharmacological inhibition of GPT2 reduces growth and migration of GBM cells. This further explains the effect of GPT2 on tumour growth and metastasis . In anottastasis .Furthermore, GPT2 cooperates with glutamine transporters to promote tumour metastasis. In a study identifying colorectal signet ring cell carcinoma (SRCC), measurement of the uptake of three nutrients, glucose, fatty acids, and glutamine, by cancer cells revealed the metabolic profile of SRCC. SRCC increase glutamine uptake and metabolism by upregulating GPT2 and SLC1A5, leading to tumour resistance and malignant progression. GPT2 and SLC1A5 inhibitors may inhibit tumour growth and distant metastasis, and increase susceptibility to 5-fu and L-OHP therapy .PIK3CA mutations increase glutamine dependence by upregulating GPT2 and reprogramming glutamine metabolism in CRC cells , a key enzyme in the intracellular synthesis of glutamine, catalyzes glutamine production using ammonium chloride and glutamic acid under an energy supply of ATP. GS expression promotes asparagine and nucleotide synthesis and enhances the uptake of essential amino acids by the extracellular region of the TME . GS can in vivo and in vitro experiments that GS could promote the metastasis of HCC cells by promoting the expression of EMT molecules [In a trial on the clinical significance of GS, it was concluded from olecules . Previouolecules ,78. In aolecules . This pholecules .3Oxidative stress refers to damage to cell DNA, proteins, and lipids caused by the excessive accumulation of intracellular ROS, which leads to cell death . Iron-reMany studies have confirmed the antioxidant effect of glutathione on tumour metastasis. For example, Liu et al. confirmed that GPX4 and GSTM1 are responsible for the high consumption of GSH and reduction of ferroptosis in lung adenocarcinoma to improve the chemical resistance of tumour cells and achieve brain metastasis of primary lung cancer cells . PiskounSLC3A2 (solution carrier family 3 member 2) is a type II transmembrane glycoprotein of the SLC3 family and is a heavy chain component of amino acid transporters (HATS). SLC3A2 is widely expressed and participates in the transport of most important amino acids. Increasing evidence has shown that the transport of amino acids through SLC3A2 plays an important role in the occurrence and progression of tumours.In a study of colorectal cancer, knockdown of MIF and SLC3A2 promoted cell iron-related death and inhibited tumour growth and metastasis via the AKT/GSK-3β pathway . SimultaSLC7A11 also plays an important role as a catalytic subunit of xCT in resisting oxidative stress, inhibiting cell iron-related death, and promoting tumour metastasis. In a study on ESCC, SLC7A11 expression was associated with lymph node metastasis in 127 patients with ESCC undergoing radical chemoradiotherapy. Patients with high expression of NRF2 and SLC7A11 had significantly shorter overall progression-free survival and poor response to treatment. SLC7A11 inhibits cell iron-related death and promotes tumour radioresistance to achieve malignant tumour progression .DKK1 can be used upstream of SLC7A11 to increase the expression of SLC7A11 in breast cancer cells and protect metastatic cancer cells from lipid peroxidation and iron-related death  reported that in the tumour microenvironment, the inherent program of cells drives immune and cancer cells to obtain glucose and glutamine, respectively. However, glutamine uptake by cancer cells can inhibit glucose uptake by normal bone marrow and immune cells, creating a microenvironment suitable for tumour growth and metastasis . Researc+ T cell infiltration and a decrease in the number of myeloid cells through a cytokine-induced increase in STAT1 and weakened STAT3 phosphorylation in T cells, causing metastatic cancer cells to exhibit interferon-gamma (IFN-γ)-responsive gene labelling and undergo apoptosis [In a study on intraperitoneal metastasis of ovarian cancer, researchers found that deletion of Tissue transglutaminase (TG2), an enzyme overexpressed in primary ovarian cancer, can delay intraperitoneal metastasis of ovarian cancer and reduce ascites. In addition, the deletion of TG2 can lead to an increase in CD8poptosis .It can ipoptosis . In a stpoptosis .Fig. 2MeStudies have shown that glutamine transporters play important roles in this process. In a study of HCC, IL-1β-induced SLC7A11 overexpression via the IL-1R1/ERK/SP1 pathway upregulated PD-L1 and CSF1 via the αKG/HIF1α axis. SLC7A11 promotes TAMs and MDSCs infiltration. The CSF1R inhibitor, BZL945, and anti-PD-L1 antibody blocked SLC7A11-induced HCC metastasis. Glutamine promotes tumour cell metastasis by affecting immune checkpoints and infiltration . Through−/− deficient mice, which lack the V(D)J recombinase mechanism necessary to rearrange the antigen receptor gene and are therefore unable to produce mature B or T lymphocytes or adaptive immune responses [In contrast, the blockade of glutamine metabolism increased endogenous antitumour immunity, even without concomitant immunosuppressants, which may be related to the increase in available glutamine content in the TME after blocking glutamine metabolism in tumour cells. When JHU083 was administered with the anti-PD-1 antibody, it showed a more significant antitumour effect than when administered alone . Notablyesponses .In 2022, Jiang et al. pointed out that in the microenvironment, tumour cells can increase the use of glutamine by overexpressing glutamine-fructose-6-phosphate transaminase 2 to inhibit the mitochondrial fission of macrophages. However, the cytoplasmic calcium increased by mitochondrial division in macrophages can eliminate the phase transformation of the Wiskott-Aldrich syndrome protein-Wiskott-Aldrich syndrome interaction protein (WIP) complex and phosphorylate WIP through protein kinase C-θ (PKC-θ), which is a vital link for tumour cells to escape from phagocytosis .in vivo. This method effectively provides T cells with glutamine while inhibiting the glutamine metabolism of tumour cells, and remarkably improves the acidic, hypoxic, and low-nutritional tumour microenvironment to reprogram tumour and immune cell metabolism, induce immunogenic cell death, promote dendritic cell maturation, reduce the number of immunosuppressed cells, trigger a strong tumour-specific immune response, and regulate the tumour immunosuppressive microenvironment [+ T cell infiltration and inhibit primary or metastatic tumours [Finally, several studies on distant metastasis caused by glutamine-promoted immune escape are underway. There is an increasing number of cases of glutamine inhibitors combined with various immune drugs to treat primary tumours and prevent distant metastasis . Xie et ironment . Furtherironment ,122. In  tumours .5In addition to glutamine-metabolising enzymes, transporters, and other metabolic factors that affect immune cell function to promote metastasis, glutamine also promotes tumour cell metastasis in rare ways.In a study on breast cancer in 2021, Torrino et al. observed that changes in the mechanical signals of the tumour microenvironment, such as enhanced tumour cell contractility, mechanical expansion of tumour blocks, and changes in the physical properties of the surrounding matrix materials, can regulate cell mechanics and affect cell metabolism to promote cancer invasion. Microtubules (MTs) have been a hot topic in recent studies of cell structure, and their biological structure determines whether they can carry large amounts of PTMs, such as acetylation, glutamylation, and glycosylation. PTMs involve the addition of a variable number of amino acid residues (e.g. glutamic acid and glycine) as secondary branches of the tubulin backbone. MTs exhibit specific cellular functions. Further studies have revealed that mechanical signals can glutamylate MTs, affect the mechanical stability of cells, and promote tumour cell metastasis, revealing the relationship between cell metabolism and MT kinetics as well as cancer invasion .Glutamine has also been shown to promote tumour growth and metastasis by activating inflammatory mediators such as platelet-activating factor (PAF) . GlutamiMestre-Farrera and colleagues show that CAFs rely much more on glutamine than epithelial tumour cells. Glutamine dependence drove CAF migration toward this amino acid when cultured in low glutamine conditions: CAFs invaded a Matrigel matrix following a glutamine concentration gradient. Stimulation of glutamine-driven epithelial tumour invasion by fibroblasts required previous CAF activation. The specific mechanism includes a TGF-β/Snail1 signal axis and a polarised Akt2 distribution modulated by the Gln-dependent TRAF6 and p62 in the migrating front .6Distant metastasis, one of the cancers with the worst prognosis, significantly reduces the quality of life of patients and shortens their survival time. This has also brought great pressure and burden to modern medical systems. However, no significant progress has been made in the prevention or treatment of cancer metastases. Chemotherapy and palliative therapy do not benefit patients with most conditions. Therefore, new treatments for cancer metastasis are required .With advances in the research field of nutrient metabolism in cancer metastasis, we have gradually realised the importance of metabolic substances in the occurrence and development of cancer. Compared to sugars and lipids, amino acids, the most flexible substances in the body, have more complex and diverse physiological functions. For example, glutamine plays an indispensable role in biosynthesis, antioxidant defense, chromatin modification/gene transcription, promotion of transmembrane transport of other amino acids, and regulation of cell signalling . Recent As the amino acid with the highest content in tumour cells, glutamine has various effects on tumour metabolism and malignant progression. As the most important component of glutamine metabolism, glutamine metabolic enzymes can affect tumour cell proliferation and metastasis through a variety of pathways, including regulating dry properties through ROS/Wnt/β-catenin signalling, promoting cell proliferation and metastasis through interaction with miRNAs, regulating the expression of tumour EMT-related molecules, and promoting AMPK activation and mTOR signalling inhibition to affect tumour cell metabolism and metastasis . The intIn this study, By searching the literature about the influence of glutamine metabolism on tumour metastasis, we refer to the previous model of other nutrients affecting tumour prognosis, and take glutamine metabolism process as the starting point to elaborate the role of various key enzymes and transporters in influencing tumour metastasis in detail. Hoping to reveal the influence of glutamine on tumour metastasis through the overall process of glutamine metabolism. It puts forward new ideas and directions for studying the influence of metabolic pathways on tumour growth and prognosis. In addition, we also hope to describe the small molecular drugs targeting tumour metastasis based on glutamine metabolising enzymes and transporters.+ T and other immune cells and reduce the number of myeloid cells. They can also work with anti-PD-1 and other immune checkpoint inhibitors to inhibit the distant metastasis of tumour cells and enhance the effect of the microenvironment on immune injury [With a continuous increase in research in this field, an increasing number of drug inhibitors of glutamine uptake and catabolism have been developed. Some of these are currently in clinical trials or have been approved by the Food and Drug Administration for use in patients with cancer or other conditions . Glutamie injury .In the future, the most effective molecular drugs for directly targeting glutamine, glutaminase, or transporter inhibitors will provide new ideas and methods for treating adverse prognoses such as tumour metastasis, which will bring substantial and effective assistance to the field of tumour cell metastasis inhibition through metabolism. In addition, they can provide patients with high drug efficacy in the clinic, thus helping to curb tumour progression . In conc10.13039/501100001809National Science Foundation of China (Grant No. 81672769) and 10.13039/501100017599Major Science and Technology Projects of Zhejiang Province (2021C03078)This study was supported by grants from Review and/or approval by an ethics committee was not needed for this study because this study does not involve ethics.The data related to this study are not stored in a publicly available repository. No data was used for the research described in the article.Xugang Zhong: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Zeju He: Data curation. Li Yin: Project administration. Yong Fan: Project administration. Yu Tong: Project administration. Yao Kang: Conceptualization, Funding acquisition. Qing Bi: Conceptualization, Resources, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


This data article describes the process of data collection and analysis of Twitter conversations about sustainable products. The dataset contains the IDs of tweets tagged with the hashtags #sustainableproducts, #ecoproducts, #ecofriendlyproducts, and #greenproducts. The time period spans 10 years and includes a total of over 140 thousand tweets from around the world. The article describes the process of obtaining the data using Twarc and the Twitter developer's academic researcher API and describes the preprocessing techniques used to identify keywords, hashtags, topics, and sentiments expressed in the conversations. The analysis identifies key attributes of each sustainable product category as well as commonalities and differences within and across categories. The data have the potential to be reused in future research related to sustainable consumption and production, including further analysis of the sentiments and attitudes expressed in the Twitter conversations and comparison with other social media platforms or survey data. In addition, the data can serve as a basis for marketing strategies and product design by enterprises or organizations seeking to promote sustainable products. Specifications Table•Our dataset is unique, providing a wider understanding of sustainable products across social media. It contains IDs for more than 140 thousand historical tweets grouped into 4 product categories labeled with the hashtags #sustainableproducts, #ecoproducts, #ecofriendlyproducts, and #greenproducts.•This dataset has the potential to attract the interest of researchers, enterprises, NGOs, and policymakers. While the characteristics of sustainable products have been studied by researchers in various fields, we anticipate that this dataset will contribute to a deeper understanding of consumption patterns on social media.•This dataset can be used for sustainable consumption and production related research, including studying characteristics over specific time periods and making comparisons with other social media platforms.1The creation of this dataset was motivated by a desire to explore the attributes of sustainable products. Since social media platforms such as Twitter provide a rich source of data on consumer attitudes and behaviors, it is imperative to investigate novel approaches for analyzing and using this data to gain valuable insights into sustainability-related consumer behavior issues. This approach enables a better understanding on the perceptions and discourse surrounding sustainable products among social media users, facilitating the identification of key themes and sentiments associated with these discussions.This dataset is used to provide insights for marketing strategies and product design aimed at promoting sustainable products. By employing this approach, business entities can gain valuable insights regarding the significance of various characteristics and attributes to consumers, as well as the manner in which these aspects are discussed within the context of social media.2Twitter is a microblogging service that allows people to share updates, news, and information within their network and beyond Twarc is a powerful Python-based tool that can help researchers and analysts gain deeper insights into Twitter data Datasets were collected via the Twitter Academic API using the open-source Python library Twarc 3The replication of this research can be done twofold: (A) replicate the methodology by retrieval of OSF repository data 3.13.1.1In accordance with the terms of use and privacy policy of Twitter developers, the sustainable products dataset only contains tweet IDs. As a prerequisite, a Twitter API developer account is needed to obtain the Twitter API credentials After hydration, each of the resulting data frames has headers, allowing for the necessary steps to engage in data processing.3.1.2As stated previously, a Twitter API developer account is needed to obtain the Twitter API credentials 3.2Preprocessing of Twitter data retrieved with either method (A) or (B) is an important step to solve the problem of noise in the data 3.3In this section, several steps are presented, as revealed in 3.3.1User analysis shows that 2,976 authors delivered a total of 5,001 #sustainableproduct tweets, 2,026 authors delivered 5,394 #ecoproduct tweets, 4,878 authors delivered 14,980 #ecofriendlyproduct tweets, and 3,620 authors delivered 8,432 #greenproduct tweets.3.3.2The use of content analysis, typically used for analyzing text documents, is a valid, rigorous, reliable, and replicable research method In addition to the term frequency analysis, we extracted all the hashtags that appeared in tweets and analyzed them in Python. Some of the most frequently occurring hashtags are presented in 3.3.3We utilized TweetNLP 3.3.4We used RoBERTa, a pre-trained language model, to perform sentiment analysis for the tweets in our study Textblob results exhibit different results, tending to balance the shares among the positive and neutral sentiment categories and increasing the shares of the negative ones. Conversations labeled with #sustainableproducts and #ecoproducts exhibit similar shares of positive tweets, while #ecofriendlyproducts have the highest share of positive tweets (52.45%). Similar to sentiment assessment done with RoBERTa, #greenproducts tweets assessed with Textblob received the greatest share of negative-sentiment tweets (17.14%) and the lowest share of positive-sentiment tweets (41.65%).4The current methodology described in this study was applied to four tweet datasets on #sustainableproducts, #ecoproducts, #ecofriendlyproducts, and #greenproducts, providing aggregated insights on content, topics, and sentiments. We anticipate the datasets hold potential for further analyses and can be expanded to explore both current and additional datasets. For example, it would be interesting to examine variations in tweet characteristics across distinct time periods and compare them across different product categories. This methodology stands for further endeavors to study sustainable production and consumption patterns on social media.5The Twitter API requires being used in order to obtain data according to the research article's strategy. However, as of March 27, 2023, Twitter deprecated the tool we employed This study was conducted in accordance with ethical guidelines and regulations. This study involved the analysis of publicly available Twitter data. No informed consent was required because all the tweets analyzed were already publicly available. We ensured that participant data was fully anonymized and that no personal information was included in the dataset. We also adhered to the Twitter platform's data sharing policies and terms of use. We obtained permission from Twitter to use the Twitter programming interface for academic research.Cristian Toșa: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. Ari K.M. Tarigan: Resources, Writing – review & editing, Supervision, Project administration.


Funding statement. We found that Funding part in the published articles was wrong in “This study was financially supported by grants from the Education Science and Technology Innovation Project of Gansu Province (grant number GSSYLXM-02) and the Fertilizer Production and Demonstration Based on Livestock and Poultry Waste in Gansu Province (grant number 2020 C-16),” which was due to our negligence in submitting the revised papers. The correct Funding statement appears below.In the published article, there was an error in the