Drp1, increased Mfn2, and decreased PGC-1, NRF1, and TFam, respectively. The mitochondrial DNA (mtDNA) copy number exhibited no change while the mitochondrial mass increased. In agreement, studies in isolated mitochondria from mouse liver also showed dexamethasone-induced reduction of mitochondrial respiratory function, as suggested by decreased mitochondrial respiration controlling rate (RCR), lower MMP, declined ATP synthesis, opening of the mitochondrial permeability transition pore (mPTP), damage of mtDNA, and the accumulation of ROS. In summary, our study suggests that mitochondrial dysfunction occurs along with dexamethasone-induced insulin resistance in 3T3 L1 adipocytes and might be a potential mechanism of dexamethasone-induced insulin resistance. Dexamethasone is a glucocorticoid analog, which is reported to induce insulin resistance and to exacerbate diabetic symptoms. In this study, we investigated the association between mitochondrial dysfunction and the pathophysiology of dexamethasone-induced insulin resistance. An insulin resistance model in 3T3-L1 adipocyte was established by 48-h treatment of 1 μM dexamethasone, followed with the detection of mitochondrial function. Results showed that dexamethasone impaired insulin-induced glucose uptake and caused mitochondrial dysfunction. Abnormality in mitochondrial function was supported by decreased intracellular ATP and mitochondrial membrane potential (MMP), increased intracellular and mitochondrial reactive oxygen species (ROS) and mtDNA damage. Mitochondrial dynamic changes and biogenesis were suggested by decreased  Insulin resistance, characterized by a dampened response to insulin signals and decreased uptake and/or utilization of glucose in the target tissue, is a major cause of type 2 diabetes ,2. DexamA large number of studies have shown that the occurrence of diabetes is closely related to mitochondrial dysfunction ,11. The Adipose tissue is one of the major targets for insulin and adipose tissue dysfunction usually leads to insulin resistance. A prospective cohort study in women found that in more than 60% of the diabetes patients their condition was related to obesity . PhosphoN-Amino)-2-Deoxyglucose (2-NBDG) uptake and protein kinase B (PKB/AKT) phosphorylation. As shown in Insulin resistance could be induced by dexamethasone in cultured 3T3-L1 adipocytes, which is widely used as an in vitro model the study of insulin resistance and type 2 diabetes mellitus . HoweverA large amount of evidence points out that the occurrence of type 2 diabetes is closely related to oxidative stress. Due to the major roles of mitochondria in both generation and elimination of cell oxidative stress. Therefore, in this study, we monitored the production of intracellular and mitochondrial ROS in dexamethasone-treated and control adipocytes using 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) and Mito-SOX probes, respectively. As shown in Cellular ATP and mitochondrial membrane potential (MMP) were important parameters in reflecting the function of mitochondria. Treatment with dexamethasone caused a marked decrease in both adenosine triphosphate (ATP) a and MMPMitochondrial mass and mtDNA, which might be altered by ROS, was also detected. As shown in PGC-1α, NRF1, and TFam, three critical factors in mitochondrial biogenesis, and Mfn1, Mfn2, and Drp1, three mitochondrial dynamic proteins, were examined with quantitative RT-PCR. The expression level of the Mfn1, Mfn2, and Drp1 was also investigated by western blot. As speculated, dexamethasone treatment caused reduced expression of PGC-1α in 3T3-L1 adipocytes. Meanwhile, NRF1 and TFam, as the downstream of PGC-1α, were also drastically decreased (Mfn2 transcription levels (p < 0.05), while Mfn1 expression was not altered. Meanwhile, levels of the Drp1 were strikingly reduced (p < 0.05). Western blot analysis of mitochondrial dynamic factors showed consistent results  is an index used to assess respiratory chain function and oxidative phosphorylation. As shown in Mitochondria are the center of energy supply in cells, providing more than 95% of ATP required for cell metabolism . Studies3T3-L1 pre-adipocytes have the ability to proliferate and differentiate into adipocytes. After being stimulated by specific inducers, they can differentiate into mature adipocytes and thus have been widely used in studies related to glucose and lipid metabolism. Many methods for establishing insulin resistance in adipocytes have been established. To study the association between mitochondrial dysfunction and insulin resistance, we established an insulin resistance model in 3T3-L1 adipocytes using dexamethasone treatment for 48 h, followed by the detection of mitochondrial function. As shown in The copy number of mtDNA in each mitochondrion was considered to be constant and thus the total copy number of mtDNA in cells tells the number of mitochondria . The repPGC-1α, NRF1, and TFam in adipocytes. After dexamethasone treatment, an insufficient energy supply, and decreased mitochondria biogenesis may work synergistically to result in a severe shortage of intracellular energy supply.Mitochondrial biogenesis is a critical adaptation of cells being exposed to chronic energy deprivation . MitochoTFam and NRF1 might underlie the maintained mtDNA levels, other unknown factors may also contribute to the compensation after dexamethasone treatment and worth further exploration.It is interesting to note that, although mitochondrial biogenesis is attenuated by dexamethasone, the mitochondrial mass is increased at the same time. A possible explanation for the discrepancy might be the impaired degradation of damaged mitochondria. Under normal conditions, harmful stimuli such as oxidative stress, aging, and energy limitation drive damaged mitochondria to be encapsulated in autophagosomes, fused with lysosomes and finally degraded. Mitochondrial autophagy abnormalities can result in increased accumulation of damaged mitochondria, leading to mitochondrial dysfunction. At the same time, due to severe mtDNA damage, the replication process of mtDNA is blocked, so the number of mitochondrial DNA remained unchanged. While a moderate reduction of In healthy cells, mitochondria undergo dynamic fusion and fission process. This dynamic process is critical for maintaining constant changes in shape, size, and network of mitochondria, which are under the control of regulatory proteins, such as Mfns and DRP1 ,27. Our 2+. The opening of mPTP may cause flow back of protons from the mitochondrial membrane space to the matrix, thereby reducing the MMP and ATP synthesis and leading to metabolic abnormalities in cells and tissues.Studies in isolated mitochondria  showed t2 at 37 °C, cells were cultured in Dulbecco’s Modified Eagle’s Medium  containing 10% fetal bovine serum  and 4.5 g/L glucose. The procession of 3T3-L1 pre-adipocytes differentiation and insulin resistance induction was performed as previously described [3T3-L1 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology . In a standard humidified incubator with a set of 5% COescribed ,29. CellFive-week-old ICR (Institute of Cancer Research) mice were provided by the Animal Center of Shandong Luye Pharmaceutical Co. Ltd.  and maintained in a breeding room of 12 h light/dark cycle, with free access to food and water. All experimental protocols involving the usage of animals were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Sciences, Northwest Institute of Plateau Biology (NWIPB20180309-02).g for 10 min, the supernatant was collected and centrifuged at 7000× g for 10 min again. Then the supernatant was discarded, and the mitochondria pellet was washed by resuspending in 5 mL isolated buffer and centrifuged twice at 7000× g for 10 min. A clean mitochondria solution was obtained after suspension in respiration buffer  to produce a mitochondria suspension with 5 mg/mL protein concentration and placed on ice for immediate use. The BCA assay was used to measure the liver mitochondrial protein concentration.As previously described , animalsTM flow cytometer  [After 24, 48, and 72 h treatment with dexamethasone, 3T3-L1 adipocytes were incubated with insulin (100 nM) for 15 min followed by incubation of 2-NBDG  for another 30min. Cells were washed twice with pre-cold PBS and suspended in 300 μL pre-cold PBS after incubation. To detect fluorescence intensity (485/20 nm for excitation and 540/20 nm for emission) for each measurement, 20,000 single cells were collected using a FACS AriaCA, USA) .Western blot analysis was performed following the manufacturer’s instruction and previous studies . After dTM flow cytometer  at 470/530 nm (ex/em) [TM flow cytometer  at wavelengths of 510/580 nm (ex/em).Production of intracellular ROS was determined by 2′,7′-dichlorofluorescein diacetate , with both confocal laser microscopy and flow cytometry. Cells were cultured on coverslips in 24-well plates and treated with 10 μM DCFH-DA at 37 °C for 30 min in a dark environment. After washing with pre-warmed PBS twice, images were captured with a confocal laser scanning microscope  at 470/530 nm (ex/em). For flow cytometry, cells were seeded in 6-well plates and treated with 10 μM DCFH-DA at 37 °C for 30 min in a dark environment. After washing with pre-warmed PBS twice, cells were suspended in PBS and analyzed with FACS Aria (ex/em) . To deteIsolated mitochondrial ROS production was also measured using DCFH-DA. Mitochondria (1 mg/mL of protein concentration) treated with 10 μM dexamethasone were incubated in 0.5 mL of the respiration buffer containing 2.5 mM succinate, 2.5 mM malic acid, 2.5 mM ADP, and 10 µM DCFH-DA for 30 min and then measured by a SpectraMax Paradigm Multi-Mode Microplate Reader  at 470/530 nm (ex/em) wavelengths.g for 5 min at 4 °C. Isolated mitochondria (1 mg/mL of protein concentration) were incubated in 0.5 mL of the respiration buffer with 2.5 mM succinate, 2.5 mM malic acid, and 2.5 mM ADP for 10 min. ATP content in cell lysates and the mitochondrial suspension was then determined using a SpectraMax Paradigm Multi-Mode Microplate Reader .ATP content in 3T3-L1 adipocytes or isolated mitochondria was measured using a luciferase-based luminescence enhanced ATP assay kit . Cells were washed twice with ice-cold PBS, harvested in 100 μL ice-cold ATP releasing buffer, and centrifuged at 12,000× TM flow cytometer  under an excitation of 488 nm [MMP was determined by JC-1 . After different treatments, cells were treated with 10 μM JC-1 at 37 °C for 30 min in a dark environment. After being washed twice with pre-warmed PBS and suspended in pre-warmed PBS, the analysis was performed by a FACS Ariaf 488 nm . IsolateTM flow cytometer  at 490/516 nm (ex/em) wavelengths [After treatment, cells were incubated with 10 μM Mito-Tracker Green at 37 °C for 30 min in a dark environment. After that, cells were washed twice with pre-warmed PBS, suspended in pre-warmed PBS, and analyzed with FACS Ariaelengths .Total genome DNA or RNA was isolated with a DNA extraction kit  or Trizol reagent , respectively. The quality of isolated DNA or RNA was determined by spectrophotometry at 260 nm. Reverse transcription was performed with 1 µg total RNA and M-MLV Reverse Transcriptase Kit . Briefly, 40 μL of total reaction volume was used in a Veriti 96 Well Thermal Cycler long PCR system  with the following reaction procedure: 3 min at 72 °C, 90 min at 42 °C, 15 min at 70 °C, and hold at 4 °C.Premix Ex Taq II), with the following procedure: 95 °C, 10 min; 95 °C, 10 s, 40 cycles, 60 °C, 15 s; 72 °C, 20 s; 72 °C, 10 min. The (2−△△Ct) value was calculated with β-actin as the inner control [The transcription level of specific genes and the mtDNA copy number were detected by RT-PCR using templates of cDNA and total genome DNA, respectively. RT-PCR was performed on a Rotor-Gene Q Sequence Detection System  using SYBR Premix Ex Taq II (Takara Bio INC) . RT-PCR  control . SequencLong PCR reactions were performed in Veriti 96 Well Thermal Cycler long PCR system using 2 pairs of primers . Long frIsolated mitochondrial respiratory was tested by measurement of oxygen consumption rates using a Clark electrode with 500 μL reaction chamber at 30 °C water bath . The cha2+-induced swelling of isolated liver mitochondria [2PO4, pH 7.4) to a final concentration of 0.5 mg/mL protein. Mitochondria solution was first treated with or without dexamethasone for 3 min at 37 °C. Then 250 μM CaCl2 was added to induce mPTP opening for 15 min. After that, absorbance was measured with a SpectraMax Paradigm Multi-Mode Microplate Reader at 540 nm .The opening of mPTP was measured by Cachondria . Isolatet-test, one-way of variance (ANOVA), using SPSS 17.0 software.All data were expressed as the mean ± SD from three independent experiments. Statistical analyses were performed using Student’s In conclusion, our results demonstrated that mitochondrial dysfunction was induced in the process of dexamethasone-induced insulin resistance. During this process, mitochondrial function was impaired, dynamics were altered, and biogenesis was inhibited. ROS overproduction led to mtDNA damage and a significant decrease of MMP followed by a reduction of intracellular ATP. To determine whether dexamethasone induced insulin resistance in 3T3-L1 cells depends on the impaired mitochondrial structure and function, and further studies are warranted.


GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm.Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner.The major seed storage protein in rice (The online version of this article (10.1007/s11103-019-00855-5) contains supplementary material, which is available to authorized users. Oryza sativa) contains two major subspecies: japonica and indica  and subsequently are translocated into the ER lumen. Prolamin is stored in protein body type-I (PB-I), which are ER-derived spherical compartments. Glutelin and α-globulin are transferred to vacuoles via the Golgi apparatus  in immature rice seeds  reporter, glutelins are believed to localize in the aleuronal and subaleuronal layers and starchy endosperm in developing rice seeds , rice mold (Aspergillus oryzae), and sake yeast (Saccharomyces cerevisiae)  brewing process. Japanese sake is a traditional alcoholic beverage in Japan, and the major ingredients used for brewing sake are polished rice , Nipponbare , Gohyakumangoku , Dewasansan , Koshihikari , Dewanosato , and Yumenokaori .Seven cultivars of brown rice were obtained from the Society for the Studies of Brewer’s Rice and harvested between 2009 and 2011. These included Five types of polyclonal anti-glutelin antibodies were generated against the following synthetic peptides: the cysteine + 210–225 amino acid sequence of GluA-2 (C + KRNPQAYRREVEEWSQ) from accession number X05664, the cysteine + 288–301 amino acid sequence of GluB-1 (C + QVQYSERQQTSSRW) from accession number X54314, the 32–48 amino acid sequence of GluC-1 (LQSPRGFRGDQDSRHQC) from accession number AB016501, the cysteine + 267–284 amino acid sequence of GluD (C + RQEEHRQYQQVQYREGQY) from accession number AY429650  and anti-ADP-glucose pyrophosphorylase (AS11 1739) antibodies produced in rabbits were purchased from Agrisera antibodies .Escherichia coli BL21 cells . Glutelin proteins were induced by the addition of isopropyl-1-thio-β-D-galactoside (IPTG).Five recombinant codon-optimized glutelin acidic subunits were synthesized  and inserted to a pET-11a vector. These included (i) 1–930 nucleotide sequence of GluA-2 from rice annotation project (RAP) locus LOC_Os10g26060, (ii) 1–918 nucleotide sequence of GluB-1 from Os02g0249900, (iii) 1–921 nucleotide sequence of GluB-4 from RAP locus LOC_Os02g16830, (iv) 1–966 nucleotide sequence of GluC-1 from RAP locus LOC_Os02g25640, and (v) the 1–867 nucleotide sequence of GluD from RAP locus LOC_Os02g15090. The generated plasmids were transformed into Rossetta-gami B One hundred grams of brown rice was polished to 90% (w/w) of the original weight using a home rice polisher . Ninety grams of the 90% polished rice was sequentially polished to 70, 50, and 30 g with the aid of a grain test mill  using an HS-4 whetstone roll#60 and a roll mesh. The rotation speed was 1800 rpm for the first 10 min to eliminate the rice embryo. Thereafter, the rotation speed was reduced to 1400 rpm. Rice powder produced at each step was sampled as 90–70%, 70–50%, 50–30% rice powder. The remaining rice grains (30% rice-polishing ratio) were pulverized using a CMT-100 auto-grinder.Rice powder (20 mg fresh weight) was suspended in 600 μL of protein extraction buffer containing 4 M urea, 6% SDS, 50 mM Tris-HCl (pH 6.8), 20% glycerol, 2% DTT, 10 μg/mL leupeptin, and 100 μg/mL PMSF. The rice powder suspension was passed through a 23-G syringe 20 times and mixed gently at 100 rpm on a bioshaker for 1 h. After flushing, an 80 μL aliquot of supernatant was mixed with 20 μL of 5 × Ling’s solubilizing buffer containing 200 mM DTT, 50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 10% SDS, and 50% sucrose. Protein was denatured at 50 °C for 15 min. One hundred microliters of 2 × urea buffer  was then added to the sample.SDS–PAGE was performed using 15% polyacrylamide gels (ATTO) with protein size markers . After separation, the proteins were stained with 0.1% Coomassie brilliant blue (CBB) R-250. The gel was destained for at least 3 h before being directly scanned using an EPSON GT 900 scanner .For immunoblot analysis, proteins were transferred from gels to 0.22-μm pore PVDF membranes (Whatman) at 12 V, 60 mA, for 30 min. The membrane was soaked in blocking buffer . After incubation with target primary antibodies (1/1000–1/8000 dilution), the membrane was washed three times with PBS-containing Tween 20. The membrane was incubated with secondary antibodies conjugated with HRP before being washed a further three times with PBS-containing Tween 20. Proteins were detected by chemical luminescence  using either an LAS 1000  or ChemiDoc™ MP imaging system (Bio-Rad). QuantityOne software ver. 4.6.9. (Bio-Rad) was used to determine the intensity of detected protein bands.Brown rice (150 g) was polished to 95% (w/w) of the original weight using a domestic rice polisher set at extremely low speed. The embryo and the aleurone layer containing the pericarp and testa fractions in the rice powder were removed from the subaleurone layer fraction by vibrating separation. The separated embryo and aleurone fractions were sampled. The remaining 95% polished rice was polished further to 90% (w/w) of the original weight using a domestic rice polisher set at low speed. After removing the embryo and pieces of broken rice, the subaleurone layer fraction was collected.Each fraction (20 mg of fresh weight) was suspended in 600 μL of the protein extraction buffer and broken using a Biomasher II device . The suspension was passed through a 23-G syringe 20 times, and gently mixed at 100 rpm on a bioshaker for 1 h. After centrifugation, an 80 μL aliquot of supernatant was mixed with 20 μL of the 5 × Ling’s solubilizing buffer. Protein was denatured at 50 °C for 15 min. Urea buffer  was then added to the sample.Yamadanishiki and Nipponbare cultivars were planted in a paddy field at the National Research Institute of Brewing . With the exception of the apex caryopsis, caryopses from primary rachis branches were sampled in the morning at 6, 8, 10, 12, 17, 22, and 27 DAF and immediately stripped of the hulls before being stored at − 80 °C until required.In 2012, Total RNA of developing rice grains was prepared from 8 to 12 grains. Developing rice grains were crushed in liquid nitrogen, after which total RNA was extracted from the resulting powders using an RNeasy Plant Mini kit (Qiagen). The quality of extracted RNA was assessed using a NanoDrop 1000 Spectrophotometer, Agilent 2100 Bioanalyzer, and Agilent RNA 6000 Nano Kit. cDNA was synthesized using a SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative real-time PCR was performed in a 20 μL volume using a TaqMan kit (Life Technologies) and an ABI PRISM 7900HT PCR system (Life Technologies). The Assay IDs (Life Technologies), including primer sets and reporter sequences for “Custom TaqMan® Gene Expression Assays,” used in quantitative real time-PCR are summarized in Supplementary Table 1. The 17S rRNA gene was used as control. Relative expression quantity for each gene was calculated by the ΔΔCt method using ABI PRISM SDS 2.1.Yamadanishiki, Nipponbare, and Gohyakumangoku (sampled in 2014 NRIB) at 17 DAF were examined with immunofluorescence microscopy. For immunofluorescence staining, seed sections (approximately 4 μm thick) embedded in paraffin were deparaffinized with xylene followed by a stepwise change of ethanol. The slides were exposed to 1% hydrogen peroxide/methanol for 30 min to block endogenous peroxidase activity before being washed with water and Tris-buffered saline (TBS). The sections were incubated with anti-glutelin antibodies (1/100 diluted) or an anti-TOC75 antibody (1/100 diluted) overnight. As secondary antibodies, we used Alexa Fluor 594 donkey anti-rat IgG (against GluB-4/5), Alexa Fluor 488 donkey anti-rabbit IgG , or Alexa Fluor 488 donkey anti-rat IgG (against GluB-4/5) at a 1:50 dilution. The sections were treated with SlowFade Gold Antifade reagent with DAPI (Molecular Probes). Immunofluorescence microscopy analysis was performed using a BX-52 microscope . Images of whole sections of rice grain were captured using a VS120 virtual slide system (OLYMPUS) and analyzed using an OlyVIA software (OLYMPUS). Tissue assignment of the immature rice grain was referred to a previous report  and OverExpress™ C43 (DE3) cells produced by Lucigen . Antibodies against GluA, GluB-1, GluB-4/5, GluC, and GluD specifically recognized each glutelin acidic subunit protein , whereas the 2010 growing season had relatively high temperatures (an average temperature of 26.2 °C during 30 days after the heading of Yamadanishiki rice) (Supplementary Table 2). The average temperature in 2011 was intermediate between 2009 and 2010. High temperatures during the grain-filling period have been reported to increase the PB-II/PB-I ratio , but no obvious difference was detected in crude protein levels between the white and non-white core Yamadanishiki rice grains by SDS-PAGE .Proteins were extracted from these powders across three different crop years, 2009, 2010, and 2011, and analyzed by SDS-PAGE . The multiple banding pattern of GluA also has been observed previously . Contrarily, in the Dewanosato cultivar, the signal intensity of GluB-1 markedly diminished with polishing as seen in GluA and GluB-4/5 . Interestingly, GluC and GluD appeared to localize throughout the endosperm  and GluD (Both Nipponbare and Yamadanishiki) was observed at 17 DAF. Based on these results, we used immature rice seeds at 17 DAF for immunofluorescence labeling assays.Furthermore, to confirm the temporal mRNA expression patterns of glutelin subfamilies, we performed quantitative real-time PCR in developing rice seeds at 6, 8, 10, 12, and 17 DAF . Maximum mRNA expression of GluA and GluB was attained around 12 DAF, in agreement with previous reports . The expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral side of grains than on the dorsal side  large and small subunits, indicating successful tissue separation Fig. . All fivjaponica rice grain cultivars across 3 years. Strong GluA (GluA-1 and GluA-2) signals were detected in the outer region of the endosperm, including the subaleurone layer. GluA also was found at lower levels in the embryo, whereas no signal was detected for other glutelin subfamilies , which showed a relatively constant level of GluB-1 expression in the starchy endosperm. The results of immunofluorescence labeling microscopy analysis of GluB-1 for Gohyakumangoku support the immunoblot data (data not shown). Previously, it has been shown that the full-length GluB-1 promoter had strong activity in the aleurone and subaleurone layers, but was weakly activated in the center of the endosperm TCA” that binds to RISBZ1, the prolamin box “TGTAAAG” that binds to RPBF, and one of the major endosperm specificity-determining motifs (the AACA motif), although it contains the ACGT motif . The difference in temporal mRNA expression patterns can be linked to the spatial patterns of glutelin expression. Glutelin mRNA expression is positively regulated by two transcription factors, Rice Prolamin Box binding Factor (RPBF) and RIce Seed Beta-Zipper (RISBZ)1 Supplementary material 2 (DOCX 4243 KB)Supplementary material 3 (DOCX 14 KB)


Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.The cell envelope of  Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named “TMM transport factor A”, TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis.Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of  Mycobacteria have a complex cell wall, which is crucial for maintaining cell integrity, protects against environmental stress, provides a barrier against access of potentially harmful molecules into the cell, and plays a critical role in mycobacterial pathogenesis. The cell wall of mycobacteria is comprised of the common bacterial cell wall glycopolymer, peptidoglycan, external to the cytoplasmic membrane, as well as an additional covalently attached glycopolymer layer comprised of arabinogalactan. Arabinogalactan bridges peptidoglycan and mycolic acids, the signature long-chain lipid of mycobacteria. Arabinogalactan-esterified mycolates likely constitute the inner leaflet of the outer membrane bilayer, with the outer leaflet comprising hydrophobically associated complex lipids, including trehalose dimycolate, sulfolipids, lipomannan, and lipoarabinomannan. This outer membrane increases both the complexity of the cell wall structure and its hydrophobicity. The enzymatic steps of the arabinogalactan and mycolate precursor biosynthesis have been well described and are the targets of several antimycobacterials, including isoniazid and ethambutol –3.–16 to C18 and C24 to C26 fatty acids. The FASII system then extends these products to produce the long meromycolate chains that are the substrates for the polyketide synthetase, Pks13. Pks13 catalyzes the final condensation step to produce α-alkyl β-ketoacids (C60 to C90) which are then acetylated and transferred to the 6 position of trehalose  system that produces Crehalose . Mono-α-rehalose . TMM cansponses 814.mycobacterial membrane protein, large) are multisubstrate transporters of the resistance-nodulation-division (RND) class that usually act as homotrimers and are exporters of molecules from the outer leaflet of the plasma membrane to or through the outer membrane. In M. tuberculosis, they include lipid and fatty acid transporters that transport virulence-associated lipids across the cell envelope. Transport is driven by downhill movement of H+ in response to the electrochemical H+ gradient  transporter that is involved in the efflux of hydrophobic small molecules from or through the periplasm of membrane . Howevermembrane . To formmembrane –33. The ytoplasm –36. HoweMycobacterium smegmatis MmpL3 (MsMmpL3) in situ, we devised a native, stringent affinity purification. MsMmpL3 was fused to a flexible linker connecting the C terminus of MmpL3 to monomeric superfolder green fluorescent protein (msfGFP) at the native chromosomal locus of MmpL3. As mmpL3 is an essential gene, the normal growth rate of this strain suggests that fusion did not disrupt the essential function of the protein. Cell membranes were collected and solubilized with the mild detergent n-dodecyl β-d-maltoside (DDM). Anti-GFP nanobodies covalently linked to a magnetic bead were incubated with detergent-solubilized membranes and then extensively washed with 0.2% DDM-containing buffer. Copurified proteins were identified via shotgun mass spectrometry , MSMEG_0250 (MmpL3), or MSMEG_0410 (MmpL10). Download Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00850-19.7TABLE S2Table S2, XLSX file, 0.1 MB.Protein identification by mass spectrometry of excised band from MSMEG_0736 (TtfA)-msfGFP GFP-Trap pulldown denoted by asterisk in Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the MSMEG_5308. This seven-bladed beta-propeller protein has a homolog in M. tuberculosis, Rv1057, which has been shown to be nonessential, although M. tuberculosis lacking Rv1057 fails to properly secrete ESAT-6 and replicated poorly in macrophages -mCitrine expression strain that uniformly labels the cell membrane . Time-la10.1128/mBio.00850-19.9MOVIE S1ttfA-targeting CRISPRi strain, grown in microfluidic plate under uninduced conditions. Imaged every hour for 16 h. YFP image shown. Exposure time, 250 ms. Bar, 2 μm. Download Movie S1, AVI file, 15.0 MB.Time-lapse microscopy of MGM6433, MalF-mCitrine Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00850-19.10MOVIE S2Movie S2, AVI file, 15.0 MB.Time-lapse microscopy of MGM6433, MalF-mCitrine Msmeg_0736-targeting CRISPRi strain, grown in microfluidic plate and induced with ATc 250 ng/ml after 1 h. Imaged every hour for 16 h. YFP image shown. Exposure time, 250 ms. Bar, 2 μm. Download Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The predicted protein for MsTtfA contains a predicted N-terminal transmembrane domain from amino acids 2 to 24, indicating that it is either a transmembrane or secreted protein. To determine the localization and topology of MsTtfA, we assessed the functionality of mCitrine fused at the N or C terminus. Marker exchange with a plasmid encoding MsTtfA-mCitrine yielded kanamycin-resistant streptomycin-sensitive transformants in similar numbers to that with pAJF792, encoding the wild-type protein indicating the C-terminal fusion is functional. In contrast, the plasmid encoding an N-terminal mCitrine fusion did not yield kanamycin-resistant streptomycin-sensitive transformants, indicating that this fusion failed to complement for essential function.https://www.dropbox.com/sh/vspwtjjd5151jo1/AADXuv-jeL7qSXJpN8m8WU3Ra?dl=0). It was previously reported that mCitrine does not fluoresce when localized in the periplasm, suggesting that the C-terminal domain of MsTtfA is localized in the cytoplasmic side of the membrane , which we demonstrated as described above is functional in M. smegmatis  MUSCLE alignment of 16 reciprocal BLAST hits of TtfA. Aligned residues shown in green. Residue ruler shown for TtfA below TtfA sequence and for the overall alignment above the alignment. (B) Alignment tree of 94 TtfA reciprocal BLAST hits. Tree based on MUSCLE alignment. Download Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00850-19.2FIG S2FIG S2, PDF file, 0.4 MB.Accumulation and localization of TtfA truncations. (A) Immunoblot of strains expressing full-length or truncations of MSMEG_0736 fused to msfGFP. Lysates of TtfA-msfGFP (MGM6829) and TtfA-msfGFP truncations . Blots probed with for GFP (top) or RNAPβ (bottom) as loading control. (B) Localization of TtfA truncations fused to GFP. Residues of TtfA fused shown to left of image. TtfA-msfGFP truncations . GFP and GFP/DIC overlay shown. Exposure times for GFP, 500 ms, 75% LED. Download Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. smegmatis  CRISPRi strain grown with no ATc  or with ATc . Download FIG S3, PDF file, 0.3 MB.Pks13 depletion leads to growth arrest by 9 h after CRISPRi induction. Growth curves of Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://www.dropbox.com/sh/vspwtjjd5151jo1/AADXuv-jeL7qSXJpN8m8WU3Ra?dl=0). This pattern is very similar to the pattern observed with TtfA-msfGFP (Movie S4). To colocalize MmpL3 and TtfA we again utilized strains coexpressing mCherry and msfGFP fusions to TtfA and MmpL3. By fluorescence microscopy, MmpL3 and TtfA strongly colocalized to cell poles and septa  depletion due to the inability of TMM to flip across the cytoplasmic membrane where antigen 85 enzymes process TMM to TDM and arabinogalactan attached mycolic acids –27. To a–epletion , attribuepletion  and B. Aepletion  and founvels see .10.1128/mBio.00850-19.4FIG S414C-acetic acid-labeled cultures of MGM6005, replete (lanes 2 to 4) or depleted of DnaK for 16 h (lanes 5 to 7) prior to labeling for 1 h with 14C-acetic acid. Lane 1 is an INH-treated replete control. (B) Graph of TDM/TMM ratios for quantitation of TMM and TDM of TLCs in panel A. Download FIG S4, PDF file, 0.4 MB.DnaK depletion does not impact TMM and TDM levels. (A) TLCs of extractable mycolic acids from Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis depleted of TtfA or MmpL3. Depletion of either TtfA or MmpL3 impaired TMM transport, with the resulting accumulation of TMM and loss of TDM  and inhibitors targeting late steps in TMM/TDM transport . Resistance mutations to SQ109, BM212, and AU1235 arise in MmpL3; however, only BM212 and AU1235 have been shown to directly inhibit MmpL3 flippase activity, and all three inhibitors may have effects outside MmpL3 flippase activity –26, 50. –MSMEG_5308 using CRISPRi and verified efficient knockdown using an MSMEG_5308-msfGFP strain (see M. smegmatis (data not shown).The identification of MSMEG_5308 as an MmpL3/TtfA interacting protein suggested that MSMEG_5308 may colocalize with the MmpL3 complex. Indeed, MSMEG_5308-msfGFP localized to cell poles and septa in a pattern similar to those of both TtfA-msfGFP and MmpL3-msfGFP by live cell fluorescence microscopy . To examrain see . Depleti10.1128/mBio.00850-19.5FIG S5FIG S5, PDF file, 0.5 MB.MSMEG_5308-msfGFP depletion using CRISPRi. Lysates from MGM6730 grown without or with ATc for 12 h and probed for GFP (top) or or RNAPβ (bottom) as loading control. Download Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assess the effect of MSMEG_5308 on MmpL3/TtfA complexes, we isolated TtfA-msfGFP using anti-GFP nanobodies and probed for MmpL3-mCherry in the presence and absence of MSMEG_5308. In DDM-solubilized lysates, TtfA copurified with MmpL3-mCherry in MSMEG_5308-depleted lysates similarly to control cells . HoweverWe have identified two new components of the essential machinery of mycolic acid transport and cell growth in mycobacteria. The MmpL3 transporter was previously known to transport trehalose monomycolate, but its cofactors were unknown. The MmpL3 machinery contains the essential protein TtfA, which we show is required for TMM flipping across the cytoplasmic membrane. A third complex member, MSMEG_5308, while not required for TMM transport, appears to stabilize the MmpL3 complex and is upregulated in response to MmpL3 dysfunction. All three of these proteins localize to cell poles and septa, which are the sites of cell elongation and the previously reported localization sites of early mycolic acid synthetic machinery such as MabA and InhA as well as MmpL3 .TtfA, a protein with no predicted domains of known function, is an essential component of the mycolic acid transport machinery. We have defined the essential portions of TtfA, amino acids 1 to 205 that include the N-terminal transmembrane domain but not the poorly conserved disordered C terminus. Using coprecipitation techniques, we see that truncations that disrupt localization and interaction with MmpL3 fail to support the essential TMM transport function of MmpL3. Our model for the molecular function of TtfA in TMM transport is that the protein links the mycolate biosynthetic machinery to the MmpL3 transporter, possibly by binding directly to TMM. MmpL3 is distinct from several other MmpL proteins in that disruption of the transporter does not inhibit synthesis of the transported lipid. For several MmpL proteins, transport and synthesis are linked. For example, deletion of the sulfolipid transporter MmpL8 abolishes sulfolipid synthesis rather than simply inhibiting its transport , 51. Sim53–M. tuberculosis, which we also find in our purifications of MsTtfA . All M. smegmatis strains were cultured in LB or 7H9 medium with 0.5% glycerol, 0.5% dextrose (LBsmeg/7H9smeg). M. tuberculosis was grown in 7H9-oleic acid-albumin-dextrose-catalase (OADC); 0.05% Tween 80 was added to all liquid media. Antibiotic concentrations used for selection of M. smegmatis and M. tuberculosis strains were as follows: kanamycin, 20 μg/ml,; hygromycin, 50 μg/ml; streptomycin, 20 μg/ml. For CRISPRi knockdowns, anhydrotetracycline (ATc) was used at 50 ng/ml (M. smegmatis) or 100 ng/ml (M. tuberculosis).Standard procedures were used to manipulate recombinant DNA and to transform f mc2155 . M. tubeelection . All str10.1128/mBio.00850-19.8TABLE S3Table S3, XLSX file, 0.1 MB.Bacterial strains, plasmids, and CRISPRi oligonucleotides used in this study. Download Copyright © 2019 Fay et al.2019Fay et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli RNAPβ monoclonal, 1:20,000; Neoclone), and Ag85  antibodies were used.For protein and epitope tag detection, GFP , mCherry , RNAP-β  adhered to a 25-mm by 75-mm glass slide. A second slide was pressed down on top, and the setup was allowed to cool at room temperature for 10 min. The top slide was removed, and the pad was cut and removed so that a 3- to 4-mm strip remained near the center. Two to three microliters of M. smegmatis culture was added to the pad, and a number 1.5 24-mm by 40-mm cover glass was sealed to the Gene Frame. Slides were incubated in a stage-top incubator at 37°C. For time-lapse microscopy, cells were incubated in CellAsic ONIX microfluidic system  at a flow of 2.0 psi and heated at 37°C. Cells were equilibrated in plates at 37°C for 3 h prior to the start of imaging. Cell lengths were quantitated using Zeiss Zen software.All images were acquired using a Zeiss Axio Observer Z1 microscope equipped with Definite focus, stage top incubator , Colibri2.0 and Illuminator HXP 120 C light sources, a Hamamatsu ORCA-Flash4.0 CMOS camera, and a Plan-Apochromat 100×/1.4 numerical aperture oil differential inference contrast (DIC) lens objective. Zeiss Zen software was used for acquisition and image export. The following filter sets and light sources were used for imaging: GFP , mCherry , yellow fluorescent protein (YFP)  and FM 4-64 . For cell staining, 100 μl of culture was used. A final concentration of 1 μg/ml FM 4-64 (Invitrogen) was added. Cells were pelleted by centrifugation at 5,000 × M. smegmatis and M. tuberculosis cultures were grown and depleted for the following times: M. smegmatis CRISPRi, 6 h in ATc 50 ng/ml; M. smegmatis Tet-DnaK, 16 h without ATc; and M. tuberculosis CRISPRi, 26 h in ATc 100 ng/ml. For TMM and TDM labeling, 1 ml of culture was removed and labeled for 1 h with 1 μl for M. smegmatis or 16 h with 2 μl for M. tuberculosis using [1-14C]acetic acid, sodium salt . For INH controls, 20 μg/ml INH was added 5 min prior to label addition. After the incubation, cells were harvested by centrifugation at 10,000 × g for 5 min, and the supernatant was removed. The pellet was resuspended in 500 μl chloroform-methanol (2:1) and incubated at 37°C for 2 h. Cells and debris were pelleted at 10,000 × g for 5 min, and the supernatant was removed. Ten microliters of chloroform-methanol extraction was spotted on high-performance thin layer chromatography (HPTLC) plates, run 3 times in chloroform-methanol-water (90:10:1), allowed to air dry, and then imaged using a Phosphor storage cassette and Typhoon Trio . ImageJ64 was used to quantitate the radioactive signal.M. smegmatis mc2155 expressing native MSMEG_0736 (MGM6425), MSMEG_0250 (MGM6464), or MSMEG_0410 (MGM6484) with a C-terminal msfGFP-tag. M. smegmatis strains were grown in 7H9 with 0.05% (vol/vol) Tween 80. Harvested cells were washed three times with phosphate-buffered saline (PBS) and frozen before lysis with a cryogenic grinder (SPEX SamplePrep). The powder was resuspended in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, SIGMAFast protease inhibitor cocktail (Sigma-Aldrich), and 0.6 to 0.7 units/ml Benzonase endonuclease, and the solution was incubated for 30 min. Solutions were centrifuged at 15,000 × g for 30 min, followed by centrifugation at 98,000 × g to 99,594 × g  for 1 h to isolate the membranes. Membranes were solubilized for 1 h at 4°C in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% DDM using a 1:0.10 (wt/wt) ratio of membranes to detergent. MSMEG_0410 was solubilized using a 1:0.15 (wt/wt) ratio of membranes to detergent. The solutions were centrifuged for 30 min at 99,526 × g to 103,530 × g. Solubilized membranes were incubated with GFP-Trap_MA beads (ChromoTek) for 1 h at 4°C. The beads were washed three times with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.2% DDM. Proteins were eluted from the beads by the addition of 0.2 M glycine (pH 2.5), and the eluate was neutralized with 1 M Tris base (pH 10.4). The elution was repeated a second time.Endogenous MSMEG_0736 (TtfA), MSMEG_0250 (MmpL3), or MSMEG_0410 (MmpL10) was purified from 18 columns (Nest Group) and then evaporated to dryness. Samples were resuspended in 0.1% formic acid for mass spectrometry analysis.The two GFP-Trap_MA elutions were pooled, and proteins were precipitated with trichloroacetic acid. The pellets were resuspended in 0.1% RapiGest SF in 50 mM ammonium bicarbonate. Samples were prepared for mass spectrometry analysis as previously described . Samples18 reverse phase capillary column . Peptides were then separated by an organic gradient from 5% to 30% acetonitrile (ACN) in 0.1% formic acid over 180 min at a flow rate of 300 nl/min. The mass spectrometer (MS) continuously collected spectra in a data-dependent fashion over the entire gradient.Samples were analyzed on a Thermo Scientific Orbitrap Fusion mass spectrometry system equipped with an Easy nLC 1200 ultrahigh-pressure liquid chromatography system interfaced via a nanoelectrospray source. Samples were injected onto a CMycobacterium smegmatis UniProt reference proteome database. Variable modifications were allowed for methionine oxidation and protein N terminus acetylation. A fixed modification was indicated for cysteine carbamidomethylation. Full trypsin specificity was required. The first search was performed with a mass accuracy of ±20 ppm, and the main search was performed with a mass accuracy of ±6 ppm. A maximum of 5 modifications were allowed per peptide. A maximum of 2 missed cleavages were allowed. The maximum charge allowed was 7+. Individual peptide mass tolerances were allowed. For MS/MS matching, a mass tolerance of 0.5 Da was allowed, and the top 6 peaks per 100 Da were analyzed. MS/MS matching was allowed for higher charge states and water and ammonia loss events. Data were searched against a concatenated database containing all sequences in both forward and reverse directions, with reverse hits indicating the false discovery rate of identifications. The data were filtered to obtain a peptide, protein, and site-level false discovery rate of 0.01. The minimum peptide length was 7 amino acids.Raw mass spectrometry data were analyzed using the MaxQuant software package (version 1.3.0.5) . Data we3 pieces and subjected to a modified in-gel trypsin digestion procedure as follows  with a flame-drawn tip  solvent A . A nanoscale reverse-phase HPLC capillary column was created by packing 2.6-μm Crawn tip . After eAs peptides eluted, they were subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer . Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest  . All dat600 of 0.5. Nontargeting and MSMEG_5308 depletion strains were grown with ATc 50 ng/ml for 24 h, and Pks13 depletion strains were grown with ATc for 6 h. Cultures were cooled on ice, and cells were harvested by centrifugation . Pellets were washed once with 1 ml of PBS. Pellets were resuspended in 500 μl PBS with 1× HALT protease (Thermo Scientific) and lysed via bead beating  2 times for 1 min with 5 min on ice between. Beads, unbroken cells, and debris were pelleted at 5,000 × g for 10 min at 4°C. The supernatant was collected, and an additional 500 μl of PBS containing either 1% DDM or 1% Triton X-100 was added and incubated at 4°C for 1 h with rocking. Insoluble material was then pelleted at 21,130 × g for 1 h at 4°C, and the supernatant (∼1 ml) was collected and added to 20 μl prewashed GFP-Trap magnetic agarose beads (Bulldog Bio) and incubated for 2 h at 4°C with rocking. After the incubation, the beads were collected with a magnet and washed 3 times with 1 ml PBS and 0.1% DDM or Triton X-100. Elution was performed using SDS sample buffer with heating at 60°C for 15 min.Fifty milliliters of LBsmeg cultures of TtfA-msfGFP truncations and MmpL3-mCherry coexpression strains with CRISPRi targeting constructs were grown to an ODData for mass spectrometry identification of interacting proteins are provided in


Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are:•No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors.•The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary.•The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method. Recipe adapted from [•Phosphate-buffered saline (PBS):2HPO4, 0.24 g/L (1.8 mM) KH2PO4, adjust to pH 7.4 with HCl8 g/L (127 mM) NaCl, 0.2 g/L (2.7 mM) KCl, 1.44 g/L (10 mM) Na•Aspirator, e.g. Bel-Art Products, Thomas Scientific, # 1185B57:or otherwise Pipett aid with 10 and 5 ml serological pipettes•1.5 ml microfuge tubes, one per sample•1.5 ml microfuge tubes, for aliquoting Denaturing Protein Sample Buffer•20–200 μl pipette, yellow tips•Cell scraper, with angled swivel blade:length ∼1.3 cm (for 6-well microtiter plate) or 2 cm (for 6 cm plastic dish).e.g. Corning # 07-200-365 (1.8 cm), or TPP, # 99002 (1.3 cm) or # 99003 (2 cm)•Table top microtube centrifuge, 4 °C and room temperature (RT)•−80 °C (preferred) or −20 °C freezer•Shaking heating block, such as Digital Shaking Drybath, Thermo Fisher Scientific, # 8880028, or Eppendorff Thermomixer C-PF-19703, or Heat/Cool Thermal Mixer, Boekel, # 2708111)Seed cells into 6 cm diameter plastic dishes, and conduct experiment as planned.2)Prior to harvesting, thaw Denaturing Protein Sample Buffer at room temperature.Ensure that the SDS becomes fully dissolved.3)At time of harvesting: Remove one plate at a time from the incubator, and place on ice.4)Holding the plate in an angle, remove as much medium as possible, using an aspirator.5)Level the plate, add 4 ml ice-cold PBS, swirl plate quickly.6)Holding the plate in an angle, remove as much liquid as possible, using an aspirator.7)Level the plate A:Fig. 1Hadd 80 μl Denaturing Protein Sample Buffer to the middle of the plate B.8)Immediately, using a cell scraper (∼3 cm long blade for 6 cm plastic dish), remove cells from the plastic surface C. For thDuring this procedure the liquid becomes viscous D.9)Holding the plate in an angle, use the scraper to collect the entire sample in the lowest part of the plate E.10)Using a 200 μl pipette, transfer sample to the bottom of a 1.5 ml microfuge tube. Pipet slowly as sample is viscous F–H.11)If sample is not in the bottom of the tube, quickly spin in a centrifuge for a few seconds, 4 °C, ∼1000 g.12)Immediately place sample on dry ice or in liquid nitrogen. If not available, transfer tube to a −80 °C or −20 °C cold metal block, to quickly freeze the sample.Step 1: Harvesting cells13)Equilibrate shaking heating block to 99 °C.14)Transfer frozen samples directly to shaking heating block.15)Incubate in mixing heating block for 10 min, at 99 °C, with maximum rpm I.Step 2: Mixing Heat treatmentThe samples are ready for denaturing gel electrophoresis. The amount of sample to be loaded on the gel depends on the abundance of the protein of interest, and may require optimization for each specific protein. See anticipated results for directions.If not used immediately, samples can be stored at −20 °C or −80 °C until analysis. Thaw and mix samples well prior to loading.6 cells were harvested in 80 μl of final sample volume. This method was optimised for adherent cells, but could also be applicable for cells in suspension.The anticipated outcome is the obtention of mammalian cell culture protein extracts suitable for quantitative western blot analysis. These extracts are generated via a cost-effective and quick lysis procedure that does not require a breaking buffer nor inhibitors, but still provide conditions that significantly reduce the risk of protein degradation and modifications that may occur during harvesting and cell lysis. In this Rapid Protein Extraction (RPE) Method, in step 1, cells were harvested by directly scraping them from the surface of the culture vessel in the presence of a Denaturing Protein Sample Buffer suitable for denaturing polyacrylamide protein gel electrophoresis (PAGE). During this procedure the sample buffer became viscous, indicative of efficient cell lysis, and the sample was easily transferrable to a microfuge tube. Step 2 arose from our discovery that heating samples at 99 °C for 10 min in a shaking heating block, fully removed the viscosity of the sample. This indicated that in addition to protein denaturation, the high temperature in combination with shaking in the presence of SDS has also sufficiently sheared the DNA. Notably, western blots generated from such samples were of high quality and quantifiable. In particular, the phosphorylated form of the protein GCN2 (GCN2-P) – usually hard to detect with conventional methods (e.g. ) – was rIn order to test our procedure and its suitability for western blotting, in particular for scoring the amount of phospho-proteins, we aimed to detect the phosphorylation levels of the lowly abundant protein kinase GCN2 and its substrate eIF2α. GCN2 is best known for its function in monitoring amino acid availability in cells. When it detects an amino acid shortage, it auto-phosphorylates, and then phosphorylates eIF2α. Elevated eIF2α-P levels activate a signaling pathway that ultimately leads to a reprogramming of the cell’s gene expression profile, to allow cells to cope with and overcome starvation .+/+ and Gcn2−/− mouse embryonic fibroblast cells (MEFs) in Dulbeccós Modified Eaglés medium (DMEM) as described previously [GCN2-P is sometimes hard to detect in western blots of cell extracts (see below), making it difficult to score for Gcn2-P levels. To determine the suitability of the RPE Method for detecting GCN2-P, we grew Gcn2eviously . The nexeviously . Cells w+/+ and Gcn2−/− MEFs were grown as above, but exposed to MG132 for 0.5, 1, and 2 h. As before, the GCN2 antibodies generated a clear signal in the samples obtained from Gcn2+/+ cells, and it was clearly visible that GCN2-P increased 1–2 h after MG132 treatment  medium, followed by harvesting, lysis, SDS-PAGE and western blotting. We found that eIF2α and eIF2α-P, and GCN2 and GCN2-P, can be readily detected B. SignalTaken together, our studies strongly suggest that the RPE Method is robust, and applicable for the quantitative detection of a large range of proteins in various cell lines.


In this review, we focused mainly on the detailed analysis of the distinct segment of s-Poly composites/organic–inorganic hybrid membranes, the relationship between composite/organic– inorganic materials, structure, and performance. The ion exchange membrane, their size distribution and interfacial adhesion between the s-Poly composites, nanofillers, and functionalized nanofillers are also discussed. The paper emphasizes the enhancement of the s-Poly composites/organic–inorganic hybrid membrane properties such as low electronic conductivity, high proton conductivity, high mechanical properties, thermal stability, and water uptake are evaluated and compared with commercially available Nafion Nevertheless, drawbacks include storage, processing for fuel cells in the automobile, transportation, and portable applications [The fossil fuel sources are important to the auxiliary environmental pollution, and climate changes exist in the worldwide which need to discover eco-friendly and renewable alternative fuels ,2. Fuel ications ,7. Thereications . Recentlications ,11,12,13®  optimization at an approximate temperature (~≤80 °C). Unfortunately, the Nafion®-based membrane electrode assemblies (MEAs) are poor, when the temperature decreases, as well as increases above 100 °C, it can affect the loss of humidity, reduce proton conductivity, electrochemical stability, and mechanical strength of the PEM at high temperatures. In this connection, to prepare a PEM based on Nafion® membrane was reinforced with varying types of modified composites. The multilayer membranes have been developed to reduce the methanol crossover without deterioration of the proton conductivity owing to higher temperature capability, and thermo oxidative and chemical stability performance [® and other polymers such as poly vinylidene fluoride (PVDF), poly 2-acrylamido-2-methyl propanesulfonic acid (AMPS), and polypyrrole, are used to increase resistance in fuel permeation. The results suggest that the additions of higher amounts of inorganic fillers are utilized for the tendency of agglomeration in inorganic and within the polymer, which lead to incompatibility between the fillers and the polymer matrix [® membrane. The review indicates significant prospects for the imminent application of direct methanol fuel cell applications and has triggered the efforts on studies of atomic layer deposition. Especially in portable applications, there are several advantages, such as storage easy, high energy density, low cost of methanol, low emission, and direct oxidation of methanol without the reforming process ,15,16. A®  type perications ,30. Moreformance ,36,37,38formance ,40,41. Fr matrix ,43,44. Ar matrix . The lon® membranes have been widely used in the applications of proton exchange membrane fuel cells (PEMFCs). However, these PEMFCs are commercially available in Nafion® membranes is their low methanol crossover, high cost, and limitations when operating at high temperatures for DMFC applications. To overcome flaws in PEMFCs, sulfonic acid groups in the polymer backbone for the improvement of the membrane’s properties such as high proton conductivity and high temperature performance at low cost [® and sPAEK/sPAEK for DMFCs. The methanol permeability, water uptake, and proton conductivity of the sulfonated blend membranes (sPAEK/Nafion® and sPAEK/sPAEK) properties were analyzed by the phase separated morphology, as depicted in ®) has the same values of sPAEK due to a rich layer thickness of sPAEK , which is 1.3 times higher than Nafion® 117 membranes. From the result, it was observed that the SPEEK/PBa blend membranes showed excellent properties, and this can be due to the cross-linking between SPEEK/PBa and the rigid backbone of PBa. A multifunctional brominated sulfonated blend membrane fabricated Liu et al. [−7 cm2 s−1) and reduced proton conductive properties at 60 °C (0.064 S cm−1). This could be attributed to the cross-linked networks  between the nucleophile phenyl rings and bromobenzyl. Further, the hydrophobic nature of BPPO’s polymeric backbone and the formation of BPPO’s spherical obstacles. Consequently, the high relative selectivity values obtained from SPEEK/BPPO membranes compared to the SPEEK membrane show the highest value of 11.98 × 104 Ss cm−3 for SP-B-20, which is more than two-times the value of Nafion® 117. This may be due to the SPEEK/BPPO membrane demonstrating a much higher cell performance at the low methanol concentration and high methanol concentration  and SPEEK70/PVDF-HFP blend membranes varied between 42.3% and 31%, 43.8–38.3%, 45.7–34.8%, and 45.8–39.9%, respectively. The authors determined that the water uptake value of SPEEK70/PVDF (Mw = 275.000) blend membranes was minimized, whereas the methanol permeability value of SPEEK70/PVDF (Mw = 275.000) blend membrane (3.13 × 10−7 cm2 s−1) was much lower than that of Nafion® 117 (1.21 × 10−6 cm2 s−1) (w: 275.000) blend membranes, concerning its high proton conductivity, permeability, chemical stability, and thermal and mechanical properties. Kim et al. [6 Ss cm−3) at 25 °C was found to be six times higher due to the decrease of proton conductivity and absence of methanol crossover and values are compared with Nafion®. Hereby, we can conclude that these sulfonated composite membranes are suitable for DMFCs applications. Moreover, the study by Unveren et al. [−1 at 80 °C and 100% RH compared with Nafion® 115 (133 mS cm−1). Nonetheless, the methanol permeability was 50% lower than Nafion® 115 at a temperature of 80 °C. These changes may be due to the addition of 10 wt % PVDF . The selectivity of results showed better performance than Nafion® 115 and values are given in co-hexafluoro propylene (SPVDF-co-HFP)/sulfonated polyaniline (SPAni). The sandwiched tri-layered PAni/PVDF-co-HFP/PAni, PAni/SPVDF-co-HFP/PAni, and SPAni/SPVDF-co-HFP/SPAni membranes showed enrichment of transport properties of coated membranes and these properties was compared to SPVDF-co-HFP, PVDF-co-HFP, Nafion®, SPVDF-co-HFP/PAni blend, SPVDF-co-HFP/SPAni blend, and PVDF-co-HFP/PAni blend membranes. The coated tri-layered sandwich membranes showed higher ion exchange capacities, proton conductivities, lower methanol permeability, selectivity, and open-circuit potentials. The 2M methanol aqueous solution at 20 °C was with the coated membranes and it showed the lower methanol permeability values as low as 9.81 × 10−11 cm2 s−1 (PAni/PVDF-co-HFP/PAni), 1.72 × 10−8 cm2 s−1 (PAni/SPVDF-co-HFP/PAni), and 5.29 × 10−8 cm2 s−1 (SPAni/SPVDF-co-HFP/SPAni), respectively. The methanol permeability values are an order of two to four magnitudes lower than the Nafion®, pristine, and blend membranes  exhibited better performance in DMFCs than Nafion® . Seden et al. [−8 cm2 s−1 and 1.3 × 10−9 cm2 s−1) respectively, and these values were compared with Nafion® 117, showing lower values of proton conductivity and methanol permeability (co-hexafluoropropylene (PVDF-HFP) filled with Cloisite 30B (C30B). The DMFCs performance was observed at an open circuit voltage (OCV) 0.79 V and a power density maximum of 55 mW cm−2. The optimized membrane observed that methanol permeability and selectivity was obtained at 1.35 × 10−7cm2s−1 and 9.63 × 104 Ss cm−3 and methanol permeability values were lower than that of Nafion® 117  with sulfonated SPEEK. The PVDF-g-PSSA copolymer was dispersed homogeneously in the SPEEK matrix with a hierarchical unique particle surface. The blend membranes (SPEEK/PVDF-g-PSSA) showed better proton conductivity, lower methanol permeability, and dimensional stability higher than commercially available Nafion® 117. Especially, with a PVDF-g-PSSA content of 5 wt %, the composite membrane exhibited the best overall membrane performance, among all the composite membranes , respectively. This could be attributed to the better compatibility and strong interactions between the PVDF-g-PSSA and SPEEK matrix. The thermal stability depends upon the water uptake, dimensional change and proton conductivity of the composite membranes. In this work, the authors express the overall performance of SPEEK/PVDF-g-PSSA membranes that are produced at low cost and with superior properties, offering great potential as PEMs for DMFCs applications. Das et al. [®) composite membrane. The composite membranes are namely SPN-0 (non-sulfonated PVDF/Nafion®), SPN-2 (2 h sulfonated PVDF/Nafion®), SPN-4 (4 h sulfonated PVDF/Nafion®), and SPN-6 (6 h sulfonated PVDF/Nafion®). The proton conductivity increased from SPN-0 to SPN-4 with the value of SPN-4 at 3.6 × 10−2 S cm−1, which is higher than the Nafion® 117. In the case of methanol permeability, SPN-4 composite membranes showed maximum methanol permeability among the all the composite membranes. Likewise, SPN-4 composite membranes exhibited higher membrane selectivity compared to Nafion® 117. The authors claimed that the composite composition exhibited better glass transition temperature, higher storage modulus, higher stress relaxation and superior DMFCs performance at high methanol feed concentration. Mondal et al. [® 117 membranes using the processes of dip-coating. The blend membrane prepared by using polybenzimidazole (PBI) and partially sulfonated polyvinylidinefluoride-co-hexafluoro propylene (SPVDF-co-HFP). The proton conductivity and methanol permeability of M2 (70/30 SPVDF-co-HFP/PBI) coated membranes were higher than M3 (50/50 SPVDF-co-HFP/PBI), M4 (30/70 SPVDF-co-HFP/PBI), and M5 (PBI)-coated systems, respectively. The results indicated further that M2 membrane showed lower proton conductivity compared to Nafion®117  at a temperature of 90 °C. It was further noted that the coated Nafion® 117 membranes were electrically efficient at high temperatures in DMFCs applications. Bagheri et al. [co-HFP, prepared by a solution casting method. The SPVDF-co-HFP exhibited the finest properties in terms of liquid uptake, swelling, thermal and mechanical stability, methanol permeability, and proton conductivity in DMFC performance. The authors found that the liquid uptake, swelling, methanol permeability, proton conductivity, and thermal and mechanical stability where the best properties, in which 20 wt % (MSSP20) blend (SPEEK/SPVDF-co-HFP) membrane exhibited outstanding performances among other blend membranes . Detailed values are given in co-HFP and SPEEK blend membranes. Nevertheless, the improved properties of methanol barrier, selectivity, swelling properties, mechanical and thermal stability of SPEEK were observed by adding SPVDF-co-HFP and MSSP20 (SPEEK/SPVDF-co-HFP) to produce blend membranes. Devi et al. [co-HFP/SPES  composite membranes increased when compared to the neat SPVDF-co-HFP  composite membranes ranged from 1.709 × 104 to 2.193 × 104 Ss cm−3, which was much higher than the Nafion® 117 (0.214 × 104 Ss cm−3) membrane. Thus, in this case, the SPVDF-co-HFP/SPES  composite membrane water uptake and proton conductivity values were much lower than Nafion 117®  . The intm et al.  studied n et al.  preparedembranes  in theirn et al.  synthesieability . Furtherion® 117 . Besidess et al.  arrangedfion®117 . It was i et al.  studied i et al.  fabricatF-co-HFP . This maion 117® . −1), SFBC-50/PWA-20 (74.15 mS cm−1), and SFBC-50/PWA-30 (105.22 mS cm−1). This enhancement of mechanical strength, water uptake, proton conductivity and oxidative properties are due to three factors: (i) an excess amount of acidic and –OH groups which acted as high surface PWA; (ii) inherent nature of molecular frameworks of PWA which hold water molecules both physically and chemically; and (iii) the establishment of highly compact pores between the SFBC-50 and PWA. A similar result was observed in the absorbed water uptake values of 14.65% (30 °C) and 60.31% (90 °C) due to high-density hydrophilic functionalities in SFBC-50/PWA-10, SFBC-50/PWA-20 except for SFBC-50/PWA-30. The authors concluded that the incorporation of PWA into the polymer matrix had optimal improvement on the composites’ properties. The sulfonated fluorinated block copolymer (SFBC) was developed by Kim et al.  in nucle−1) and SPEEK-30%PVB (IEC = 2.05 meq g−1) exhibited reasonably good properties in terms of proton conductivity, mechanical stability, and methanol permeability. The authors further observed that these membranes possess tremendous properties of mechanical and methanol barriers, even though SPEEK shows very low proton conductivities. Furthermore, a study by Lin and Wang et al. [® (N/VA-b) blend membrane prepared using the casting method in the applications of DMFCs. The methanol permeability, conductivity, and performances of DMFC cell membranes were investigated and compared with Nafion® 117 and Nafion® 212. The results indicated that the blend membranes with a thickness of 50 μm have an optimum PVA content of 10 wt % and 5 wt % for both N/VA-f and N/VA-b membranes, respectively. The single cell tests were demonstrated further in the DMFCs’ performance, which showed the decreased sequence given as N/VA-f > N/VA-b > Nafion® 117 > Nafion® 212. This leads to the N/VA-f having a higher proton conductivity optimum PVA content and better DMFC performance than the N/VA-b membrane. Another study by Yang et al. [−5 cm2 s−1) when compared to S40 (PEEK sulfonated 40 h), and the lowest value of 5.592 × 10−6 cm2 s−1 in pure SPEEK membranes. In this case of SP72 (PVA:50 wt %), it decreased to 3.588 × 10−6 cm2 s−1, much lower than S40. Owing to the addition of PVA, the membrane brings an outstanding methanol resistance. In addition, the authors indicated that the slightly ion exchange capacity (IEC) and proton conductivity of the composite membranes was due to the specific interaction between SPEEK and PVA molecules. Therefore, from the above mentioned comprehensive explanations, we can conclude that the lower methanol permeability of the composite membranes suggests its potential applications in DMFC. Bhat et al. [−2 was observed in DMFCs at 50% degree of sulfonation/10 wt % mordenite phase in the PVA/PSSA composite membrane electrolytes. In addition, the methanol crossover current at low 7.5 mA cm−2 with 2 M methanol feeds on the DMFCs anode was observed further and shown in ® 117/PVA–PSSA membranes in DMFCs. Madaeni et al. [® 117 membranes. Furthermore, the homogeneous structure of the composite membranes showed better melting temperature in DSC and the enhancement of thermal stability due to the addition of PES/PVA to the PVA matrix. Yang et al. [−1, was higher than the modified PVA membranes. They concluded that the effect of cross-linking time on methanol permeability was very significant. It was seen that the selective values of the PVASA82-GA60 membrane at 21.50 × 103 Ss cm−3 and a maximum power density of 20.7 mW cm−2 were achieved at Ep,max = 0.232 V with a peak current density  of 89.20 mA cm−2 at 30 °C for the direct methanol fuel cell. To enhance membrane performance, the cross-linking of polymers and compositing of poly vinyl alcohol (PVA) has been used to achieve excellency in forming super hydrophilic membranes, and oxidative and hydrolytic stability, to overcome flaws in membrane properties ,77,78. Mg et al.  observedg et al.  observedt et al.  examinedi et al.  studied g et al.  develope−4 S cm−1) indicated that the higher proton conductivity in comparison with a dense membrane. Further, the incorporation of the nanofiber mats to the SPEEK-35%PVA matrix provided the enhanced properties of the methanol barrier and proton conductivity with a cross-linking temperature at 120 °C  exhibited the low methanol permeability (8.83 × 10−7 cm2 s−1), high tensile strength (51.2 MPa), high proton conductivity (0.084 S cm−1 at 25 °C), and high power density  solution to form a polymer electrolyte membrane, and then the alkaline uptake and swelling ratio in the thickness (LSW) and plane direction (ASW) in membranes were measured, respectively. The ionic conductivity and methanol permeability of the membranes showed that the value of methanol permeability through the membrane was lower than that of the Nafion® membrane. From the authors’ experimental data, it can be concluded that when compared to Nafion® and other studies on PCS91-glutaraldehyde (GA) membranes with higher selectivity, these membranes suggest their potential applications in DMFCs. Xiang et al. [−6 cm2 s−1 when compared to pure chitosan at 4.7 × 10−7 cm2 s−1. The content of cross-linked membrane chitosan sulfate around at ∼9.1 wt % chitosan sulfate composite membranes (CCSM 110) showed the best performance on conductivity, the proton conductivity of CCSM 110 enhanced 0.03 S cm−1 at 80 °C. Accordingly, the methanol permeability (CCSM10) was much lower than Nafion® 112 (1.9 × 10−6 cm2 s−1). In addition, the thermal analyses of cross-linked sulfate membranes were stable at below 100 °C. It concluded that the CCSM 110 with low methanol permeability is a very important part of DMFCs. Yang and Chiu  reported−7 cm2 s−1) and higher proton conductivity (6.4 × 10−3 S cm−1) when compared with the SPEEK membrane. The authors mentioned that the transportation of proton domains and increasing water uptake were contributing to the decreased permeability of methanol. Though the authors noted that the thermal and mechanical stability of the composite membrane was slightly reduced when compared to the SPEEK membrane, it was higher than Nafion® 112 membranes. Norddin et al. [3 groups, which are contributed to the surface morphology. The SPEEK membranes showed that the hydrophilic nature of top surfaces is higher than that of the bottom surface, which is indicated by the angle of the water contact and stronger migration of cSMMs. The nodule structure on the membrane are shown in Organic compounds generally contain one or more carbon atoms and covalently bond with nitrogen, hydrogen, and oxygen. The polybenximidazole, polyphosphazenes etc., are commonly used polymers in DMFC applications ,93,94. Tn et al.  discusse3 groups via cSMMs. Krishnan et al. [−1) was lower than Nafion® 115 (9.15 kJ mol−1). Furthermore, the selectivity of the blend membranes, a compromise between proton conductivity and methanol permeability, was much higher than Nafion® 115 at 15 wt % of the sulfonated cyclodextrin. The DMFCs polarizations were 2.5 M and 8.0 M methanol solutions with the variety of weight percentage  of sulfonated cyclodextrin blend membranes were determined, respectively. The blend membrane with 20 wt % of sulfonated cyclodextrin showed the highest power density of 29.52 mW cm−2 at 120 mA cm−2 and 8.0 M methanol solution. The analysis suggests that the SPEEK membranes with the addition of sulfonated cyclodextrin in DMFCs can be used as potential usage applications. Jithunsa et al. [® 115. Accordingly, the cross-linked network exhibited higher mechanical strength and better chemical stability, demonstrating the PA-CBrSPIBI membrane possibly being used as PEMs.The PEG200-HBS membrane of the smallest nodule size indicated the lowest water uptake and methanol permeability, whereas the largest nodule size indicated the highest conductivity and water uptake. The author concluded that the enhancement of proton conductivity was mainly due to the capability of the membrane which can absorb more water rather than the introduction of SOn et al.  examinedn et al.  observeda et al.  developea et al.  fabricat2, ZrO2, and TiO2) into polymer membrane can improve the thermal stability, mechanical properties, and proton conductivity at relatively high temperature (90–120 °C) [Chemical degradation of membranes is a major issue for fuel cell performance. In order to improve the long-term stability, the introduction of some inorganic compounds into the polymer composite membranes was considered as an alternative approach. The incorporation of inorganic fillers (SiO–120 °C) ,103,104.–120 °C)  examined5 Ss cm−3, which is a 123 higher than Nafion® 117 and 3.6 times higher than the pristine SPEKES are presented in ® 117 (in ® 117 c. Presum® 115 (0%) were achieved. Shabanikia et al. [3 nanoparticles with polybenzimidazole (PBI) by solution casting technique. Proton conductivity increases with a filling of nanoparticles up to 8 wt % (PSC8). This may be possible by the hygroscopic nature of SrCeO3 nanoparticles in the membrane. At higher loads (>8 wt %) of SrCeO3 nanoparticles, the proton conductivity (PSC16) decreases due to self-aggregate of SrCeO3 nanoparticles. Moreover, the nanocomposites membrane showed a greater acid uptake, high thermal stability, and proton conductivity other than the PBI membrane. Bonis et al. [2-PhSO3H) and introduced the filler of (Nafion®) developed a N_X  into the TiO2-PhSO3H membrane-based composite membrane for DMFC application. The conductivity and methanol permeability of the N_10 wt % TiO2-PhSO3H membrane showed the highest value of 0.11 S cm−1 at 140 °C. Accordingly, methanol permeability (N_10 wt % TiO2-PhSO3H) 20% reduction of limJ and better than Nafion®. This is due to the chemical nature of the surface functionalization of TiO2-PhSO3H with enhanced DMFC’s performance. Pandey et al. [® 117 and 115. The water uptake Si-PWA/PVDF membrane displayed higher value than Nafion® 117. The author exposed that the proton conductivity and methanol uptake of Si-PWA/PVDF membrane increases with increasing temperature. This is attributed to a smaller thickness of Si-PWA/PVDF membrane compared to Nafion® 117. Pandey and Shukla et al. [®117 at 25 °C and 60% relative humidity. Zhong et al. [5 Ss cm−3 compared to 1.9 times pure membranes and 8.9 times of Nafion® 117. The obtained results indicated that the cross-linked multilayer membrane structures are promising materials for DMFC applications. Ahmad et al. [® using a casting method with dimethylacetamide. The dispersion of zirconium phosphate can decrease proton conductivity, methanol permeability, and selectivity up to 5 wt % when compared to 1 wt % (zirconium phosphate). It shows better performance than that of Nafion® 117  composite 12 wt % membrane showed the best performance of proton conductivity and water uptake compared with neat SFBC-50 and Nafion® 117  compared to the neat SFBC and Nafion® 117 membranes. Han et al. [2-8 (6.69 × 10−7 cm2 S−1) and C-SPAEKS/SiO2-8 (6.71 × 10−7 cm2 S−1) at 60 °C temperature owing to SiO2 particles act as a barrier to hinder the penetration of methanol. The cross-linked structure formed in the C-SPAEKS/KSiO2 membrane can block the penetration of methanol. Further, the proton conductivity of C-SPAEKS/KSiO2-8 (0.110 S cm−1) was greater than that of C-SPAEKS/SiO2-8 (0.082 S cm−1) when compared with the Nafion® 117 (0.028 S cm−1) at the temperature of 120 °C observed and the details of conductivity values are given in ®. The obtained results were that nanocomposite membranes (NM)/GO-0.05 and 0.10  and membrane-5 (4.10 × 10−2 S cm−1 at 30 °C) (3N4) nanomaterial fabrication with SPEEK. The SPEEK/CN-0.2 nanocomposites membrane showed the highest proton conductivity  and SPEEK/CN-2.5 (4.42 × 10−7 cm−2 S−1). These changes are attributed to following issues (i) 2D g-C3N4 consisted of repeat triangular nanopores; and (ii) lower glass transition temperatures because of the mobility of the polymer (SPEEK) chains. The water uptake increases with increasing g-C3N4 nanosheets. The higher level contents of g-C3N4 nanosheets were unable to absorb excess water because the g-C3N4 nanosheets strongly suppressed the mobility of the SPEEK macromolecule. It concluded that these membranes exhibited superior thermal and mechanical properties and can be used for high-performance fuel cell application. It is crucial that the organic filler should disperse uniformly within the polymer network and minimize the self–aggregation of inorganic additives in the polymer/inorganic membranes. Moreover, inorganic fillers should not leach out from the membrane matrix during long-time applications ,115. Oneion® 117 . Furthern et al.  synthesiand 0.10  showed hg et al.  investigt 30 °C) . Besidesuctivity a among a® 117, respectively  at 130 °C/100% RH, which indicated that the increasing content of phosphonic acid act as a proton carrier. In addition, the TMSMS/FC6H4VPA membrane exhibited good thermal stability and mechanical properties. The hybrid TMSMS/FC6H4VPA membranes supporting the growth of PEFCS can cover a board operating condition from low moisture to 100% RH. Prapainainar et al. [®/MOR composites by solution casting process. The proton conductivity of the composite membrane MOR/MPTES displayed greater proton conductivity than MOR/GMPTS, and recast Nafion®. The proton conductivity of MOR/GMPTS membrane slightly decreases due to the increase in the amount of mordenite and the agglomeration processes in the mordenite of nanoparticles. Further, the increasing proton conductivity trend was absorbed in MOR/MPTES due to the increase concentrations of zeolite and sulfonic groups. Similar results were found in water uptake properties of MOR/MPTES. The higher water adsorption capacity is owing to the presence of a sulfonic group that acts as a hydrophilic group. Furthermore, GMPTS presented a higher methanol permeability compared to MPTES. This could be MPTES with good adhesion properties than GMPTS. The author found that the MOR/MPTES exposed a remarkable reduction of methanol permeability (5 wt %) and lower than MOR/GMPTS. Ahn et al. [−7 cm2 s−1) permeability and higher proton conductivity (0.0278 S cm−1) compared with Nafion® 117 . Zhong et al. [® 117. Nonetheless, the selectivity values of PVA/PMAV membrane showed an increasing and followed by a decrease of selectivity with further increasing PMAV content. Further, the authors claimed that the PVA/PMAV membrane has a low cost, admirable methanol barrier, high selectivity; good thermal stability may be a suitable electrolyte for DMFCs. Pan et al. [−3 to 10−2 S cm−1 and the optimization depends on the sulfonated polyimides containing benzimidazole group (SPIBI) degree. Wu et al. [2 functionalization using four types of amino acids  with sulfonated poly ether ether ketone (SPEEK). From the data, the water uptake and methanol permeability slightly decreased upon addition of filler content . Because of the reduction of the ionic channel size, the membrane with 15 wt % additive exhibited the best performance owing to the more hydrophilic character of hydroxyl groups. In this case, proton conductivity increased following the order SPEEK/TiO2–Scys > SPEEK/TiO2–Pser > SPEEK/TiO2–Asp > SPEEK/TiO2–H is, due to the different proton acceptor-donor capability of amino acids. The authors concluded that the high-performance of hybrid PEMs with different amino acid functionalized TiO2 fillers tunes the characters of channels/pathways of proton conduction and methanol diffusion. Chen et al. [2-P2O5 and PMMA-SiO2-P2O5-ZrO2 hybrid membranes developed by the sol-gel method. For all hybrid membranes, the proton conductivity increased with increasing temperature from 30 °C to 90 °C. For example, the hybrid membranes such as 60PMMA-30SiO2-10P2O5 and 60PMMA-30SiO2-P2O5- ZrO2 showed lower proton conductivity by the loading of ZrO2 concentration. These changes can be the processes of surface and bulk transport of the hybrid membrane. In the case of water uptake, increases with decreasing content of SiO2, this could be higher hydrophilicity of phosphate other than silicate. The authors claimed that this hybrid membrane could be applicable for low-temperature fuel cells. Ren et al. [2 hybrid membranes by then reaction 3-isocyanatopropyl) triethoxysilane (ICPTES) with tetraethoxysilane (TEOS). A series of novel organic–inorganic hybrid proton exchange membranes were prepared from sulfonated poly arylene ether sulfone Am-SPAES/SiO2 hybrid membranes showed that the proton conductivities, methanol permeability and water uptake increase with increasing the content of SiO2 . Nevertheless (Am-SPAES/10 wt % SiO2), decreases when compared with the other systems and Nafion® 117 due to the dilution effect of the excess amount of SiO2. Presumably, the improvement of thermal and mechanical stability of Am-SPAES/SiO2 hybrid membranes is attributed by the introduction of SiO2 particles. Hattori et al.  synthesir et al.  fabricatn et al.  preparedion® 117 . Moreoveion® 117  due to tg et al.  analyzedu et al.  reportedn et al.  synthesin et al.  fabricat−1 (90 °C) and maximum proton conductivity was observed SPES/PWA-30 (116 mS cm−1 at 90 °C) which is similarly related to Nafion® 117 (130 mS cm−1). The water uptake of SPES/PWA (10–30%) composite membranes decreased up to 10% and 20% other than 30% owing to the strong hydrogen bonding interaction between PWA and SPES. Mohanraj et al. [3H groups with an increase of the GO number of directional hydrogen bonds (H-bonds). The peak proton conductivity was observed at 90 °C attained by the SPEEK was 68 mS cm−1, a ternary hybrid was at 122 mS cm−1, 1.7 times better conductivity. A similar study by Mohanraj et al. [3O4-FGO) membrane in the applications of high temperature and low humidity fuel cells. The hybrid Fe3O4-FGO improves proton conductivity, water absorption, and ion exchange capacity of the membrane by increasing the number of SO3H to retaining the stable dimension. Further, the results indicated that the peak proton conductivity of SPFSGF-5 the membrane at RH of 11.13 mS cm−1, and the temperature is about 120 °C. These values are 1.44-fold better than the SP membrane; whereas the Nafion® 112 membrane exhibited a peak of proton conductivity at 9.78 mS cm−1 which was 1.13-fold lower. The authors noticed that the SPEEK/PWA and SPEEK/SPVdF-HFP/GO membranes are favorable for potential application in PEMFCs. Kim et al.  studied j et al.  enhancedj et al.  studied ® at high temperatures and low humidity conditions. However, at high degrees, s-Poly may reduce the performance of PEM in DMFCs. Hence, several functionalization and modifications have been identified for rectification of the drawbacks in polymer blends. The modifications of polymer blends are: (i) a sandwiching polymer membranes technique; (ii) the optimization of lowering precious metal requirements for the improvement of power density; (iii) electrospinning processes for the improvement of DMFC performance; (iv) long-term operation and durability; and (v) the utilization of novel polymer blends and fillers along with the combinations of inventive layering techniques which could improve the cell membrane development in the field of high temperature fuel cell applications. These future aspects can then improve the DMFCs’ performance. Various different s-Poly composites based on organic, inorganic, and organic–inorganic hybrid membranes for polymer electrolyte membranes have been reported extensively for use in direct methanol fuel cells to overcome the barrier issues encountered by Nafion® membranes. The use of suitable compatibilizer and its optimum amount can result in better high proton conductivity, low methanol permeability, high selectivity, and thermal stability and mechanical properties from a vast array of literature. Most of the literature in this review reported that the s-Poly composite membranes, such as SPEEK, SPVdF, SPES, SPAES, SEBS, sPAEK, s-PSF, and S-PEKES are promising for direct methanol fuel cell applications. Especially, these s-Poly composites and organic–inorganic hybrid membranes provide increasing proton conductivity, selectivity, thermal stability, and mechanical properties and a decreasing methanol permeability compared to conventional Nafion® membranes. An analysis of the literature shows that methanol permeability, proton conductivity and selectivity properties are the most significant in DMFCs applications. However, these properties cannot be used to predict with accuracy in the potential usage of a novel polymer. This might be due to the different aspects of stoichiometry and large oxygen in the presence of air at the cathode which can cause significant loss in fuel cell energy. Furthermore, this extensive review shows that the s-Poly composite and organic–inorganic hybrid membranes are potentially suitable for fuel cells applications at temperatures above 80–100 °C. Based on the data of the review, it can be concluded that the s-Poly composite and organic–inorganic hybrid membranes will further promote fuel cell related technology in future scenarios.We have hereby reviewed the present and future potential application of s-Poly-based composite and organic–inorganic hybrid material in order to improve the properties of PEM for direct methanol fuel cell applications. In this review, we comprehensively analyzed the chemical stability, proton conductivity, methanol permeability, selectivity, thermal and mechanical strength of different type of s-Poly composites and organic–inorganic hybrid membranes compared to commercially available Nafion


Chlorococcum sp. (UTEX B 3056). After post-incubation with fluridone, phytoene levels were measured at ~33 ug/mg cell tissue, as opposed to non-detectable levels in control cultures. Hence, the novel microalga Chlorococcum sp. is a viable candidate for the production of the high-value carotenoid phytoene and subsequent applications in cosmeceuticals, as well as more obvious nutraceutical and pharmaceutical applications.Carotenoids are lipophilic pigments found in plants and algae, as well as some bacteria, archaea, and fungi that serve two functions—(1) as light harvesting molecules—primary carotenoids, and (2) as antioxidants, acting against reactive oxygen species–secondary carotenoids. Because of their strong antioxidant properties, they are also valuable for the development of anti-aging and photo-protective cosmetic applications. Of particular interest is the carotenoid phytoene, for its colorless and UV absorption characteristics. In this study, we targeted a reduction of phytoene desaturase (PDS) activity with the pigment-inhibiting herbicide 1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]pyridin-4-one (fluridone), which leads to the over-accumulation of phytoene in the recently characterized microalgal strain  Microalgae are known to be potential sources of natural products, abundant and versatile in their activity and applications. Of particular importance are the lipophilic pigments, carotenoids. Commonly used in the food and nutraceuticals industry as colorants and dietary supplements, carotenoids have received growing popularity in cosmetics in large part, due to their antioxidant properties ,2,3,4. SHaematococcus pluvialis pyridazin-3-one) and fluridone (1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]pyridine-4-one) resistance mechanisms and mutagenesis, as well as herbicide inhibition activity . Not. NotChlocontent) . The ovecontent)  [7]. Thecontent) . Althougcontent) . A two w panel D , as therChlorococcum sp. incubated with 152 µM and 304 µM fluridone revealed the accumulation of phytoene at approximately 33 μg/mg of phytoene per dry cell weight when harvested on day four, as well as a reduction in downstream carotenoid production at both concentrations  in fluridone treated cultures, as opposed to control cultures, which showed fuller/more uniform chlorophyll fluorescence throughout the cell  and morphologically , and a final elongation step at 57 °C (10 min). Amplicons were checked for size verification and specificity by gel electrophoresis on a 1% agarose gel. The amplicons were purified from gels using an UltraClean GelSpin® DNA extraction kit  for subsequent forward and reverse sequencing  at the Functional Biosciences laboratory . Sequence data was analyzed and assembled using Geneious® . The consensus sequences were subjected to standard nucleotide similarity searches via BLASTn [Molecular characterization and identification were performed on the genomic DNA extracted from a BLASTn  against Concentrated suspensions of three series of preparation: (1) fresh, (2) 2.5% glutaraldehdye-fixed  and (3) fixed-Nile Red  (5 micrograms/mL) treated cells were deposited onto the coverslip areas in glass bottom microwell dishes  and examined by confocal microscopy using a model TCS SP5 system coupled to a DMI 6000 inverted microscope equipped with a 100× objective lens  in the x,y,z imaging mode and fluorescence scanning mode with excitation from the 488 nm line of an Argon laser. Images were collected in data sets of two channels (500–550 nm and 660–720 nm) for fresh- and glutaraldehyde-fixed cells or in three channels for Nile Red-treated cell suspensions  and examined as maximum projections (8–12 micrometers deep) in separate and graphically overlaid image channels. Fluorescence emission scans were performed from 500–750 nm using a 15 nm detector window and frame averaging of selected focal planes. DIC images were taken using the same method for laser-scanning confocal microscopy; however, the transmitted light channel  was employed here.Chlorococcum sp. culture was added to two individual 96 well microplates from BRAND® GmbH & Co. KG  at an optical density (OD) of 0.1, 250 µL per well; adapted from Franz, et al., 2013 [−1m−2), CO2 (1%), temperature (24 °C). Plates were read at wavelengths 750 nm (biomass), 680 nm (chlorophyll), and 450 nm (carotenoids) over a ten day period using a SPECTRAmax microplate spectrophotometer, Molecular Devices .A fluridone standard obtained from Sigma-Aldrich, Inc.  was dissolved in 100% ethanol and diluted accordingly: 304, 152, 76, 38, 19, 0 µM. l., 2013 . FluridoChlorococcum sp. was added to twelve sterile BD Falcon™ Tissue Culture Flasks with vented caps from BD Biosciences  at 25 mL per flask, OD 0.5. A total of six flasks were incubated with fluridone at 152 and 304 µM, respectively and subjected to the following two conditions: (1) light (22 µE per s−1m−2), CO2 (1%), temperature (24 °C); (2) high light , CO2 (1%), temperature (24 °C). Controls did not contain fluridone. Experiment was performed in biological replicates of three. UV spectrophotometric readings were taken daily on a SPECTRAmax microplate spectrophotometer, Molecular Devices , for a total of four days.® HPLC  with a 996 photodiode array detector and YMC America carotenoid column , using the YMC MTBE carotenoid method [Phytoene Profile–Algae was collected by centrifugation at 10,000 rpm and lyophilized on a Labconco FreeZone 6  for a minimum of 24h. Approximately 15 mg of algae was milled with 0.5 mm dia. zirconia/silica beads  using a Mini Bead Beater™  for a total of 2 min to achieve cell lyses. HPLC grade acetone  was added to the samples in a 1:30, mg: μL, ratio and allowed to sit for 20 min., followed by centrifugation for 10 min at 13,000 rpm and the supernatant removed and collected in a separate vial. The extraction was repeated a second time to ensure complete pigment-tissue extraction, and the supernatants combined. Samples were analyzed on a Waters 2695 Allianced method . Phytoen−1 to 175 °C and maintained for 1 min., then ramped to 235 °C at 4 °C for a total run time of 21.83 min. The instrument was tuned with a standard auto tune method and a calibration curve prepared from a Supelco 37 Component FAME mix (10 mg mL−1) in methylene chloride product # CRM47885 . The mass spectrometer operated at 70 eV in electron ionization (EI) mode with 5 scans per second between the mass range 40 and 500.Fatty Acid Methyl Ester (FAME) profiles were obtained for each treatment group via base catalyzed transesterification. 2 mL of KOH in methanol (2N) was applied to dried, ground tissue (~5 mg), vortexed and incubated at ~37 °C for 30 min. Samples were allowed to cool for approximately 15 min. 1 mL of acetic acid (1M) was added to samples to quench the reaction. Subsequently, 2 mL of HPLC grade hexane with C23:0 ISTD at 50 ppm was added to samples and vortexed thoroughly. All reagents for FAME extraction were obtained from . 200 μL of the upper portion of the sample was removed and dispensed into GC vials, fitted with inserts, for analysis. Samples were then analyzed by GC/MS on a Varian 3800 Gas Chromatograph with a Varian 2000 Mass Spectrometer and a Varian 8200 Auto sampler . 2 μL were injected onto a 30 m × 0.25 mm diam. ×0.25 µm film DB-23 capillary column  with Helium carrier gas at 1 mL/min with a 5:1 split. The inlet and transfer line were held at 250 °C. The column temperature was held at 60 °C for 1 min. and then ramped at 30 °C minStatistics were performed using Sigma Plot V11.0 . All data sets were run as a two-way analysis of variance (ANOVA), and using the Holm-Sidak method. Microplate bioassay data was run as a two-way ANOVA with repeated measures.