Identification of MIMAS, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria.

The mitochondrial inner membrane plays central roles in bioenergetics and metabolism and contains several established membrane protein complexes. Here, we report the identification of a mega-complex of the inner membrane, termed mitochondrial multifunctional assembly (MIMAS). Its large size of 3 MDa explains why MIMAS has escaped detection in the analysis of mitochondria so far. MIMAS combines proteins of diverse functions from respiratory chain assembly to metabolite transport, dehydrogenases, and lipid biosynthesis but not the large established supercomplexes of the respiratory chain, ATP synthase, or prohibitin scaffold. MIMAS integrity depends on the non-bilayer phospholipid phosphatidylethanolamine, in contrast to respiratory supercomplexes whose stability depends on cardiolipin. Our findings suggest that MIMAS forms a protein-lipid mega-assembly in the mitochondrial inner membrane that integrates respiratory biogenesis and metabolic processes in a multifunctional platform.

Isolation of intact and active FoF1 ATP synthase using a FLAG-tagged subunit from the cyanobacterium Synechocystis sp. PCC 6803.

The FoF1 ATP synthase (ATPase) is one of the most important protein complexes in energy metabolism. The isolation of functional ATPase complexes is fundamental to address questions about its assembly, regulation, and functions. This protocol describes the purification of intact and active ATPase from the model cyanobacterium Synechocystis sp. PCC 6803. Basis for purification is a 3×FLAG tag fused to the beta subunit. The ATPase is enzymatically active and its purity is demonstrated using mass spectrometry, denaturing, and blue-native PAGE. For complete details on the use and execution of this protocol, please refer to Song et al. (2022).

Expression of the Cyanobacterial FoF1 ATP Synthase Regulator AtpΘ Depends on Small DNA-Binding Proteins and Differential mRNA Stability.

FoF1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. AtpΘ prevents the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5'UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2, and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status. IMPORTANCE FoF1 ATP synthases produce ATP, the universal biological energy source. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis, but they cannot use the same mechanism as plant chloroplasts to inhibit ATP synthases during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small protein encoded by the gene atpT in cyanobacteria that can prevent the ATP synthase reverse reaction (ATPase activity). Here we found that three transcription factors contribute to the regulation of atpT expression. However, the control of mRNA stability was identified as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based regulatory mechanism within the context of the cellular redox and energy balance.

AtpΘ is an inhibitor of F0F1 ATP synthase to arrest ATP hydrolysis during low-energy conditions in cyanobacteria.

Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein AtpΘ, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of AtpΘ becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of AtpΘ to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far western blots identified subunits of ATP synthase as interacting partners of AtpΘ. ATP hydrolysis assays with isolated membrane fractions, as well as purified ATP synthase complexes, demonstrated that AtpΘ inhibits ATPase activity in a dose-dependent manner similar to the F0F1-ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways.

Genome-wide identification and characterization of Fur-binding sites in the cyanobacteria Synechocystis sp. PCC 6803 and PCC 6714.

The Ferric uptake regulator (Fur) is crucial to both pathogenic and non-pathogenic bacteria for the maintenance of iron homeostasis as well as the defence against reactive oxygen species. Based on datasets from the genome-wide mapping of transcriptional start sites and transcriptome data, we identified a high confidence regulon controlled by Fur for the model cyanobacterium Synechocystis sp. PCC 6803 and its close relative, strain 6714, based on the conserved strong iron starvation response and Fur-binding site occurrence. This regulon comprises 33 protein-coding genes and the sRNA IsaR1 that are under the control of 16 or 14 individual promoters in strains 6803 and 6714, respectively. The associated gene functions are mostly restricted to transporters and enzymes involved in the uptake and storage of iron ions, with few exceptions or unknown functional relevance. Within the isiABC operon, we identified a previously neglected gene encoding a small cysteine-rich protein, which we suggest calling, IsiE. The regulation of iron uptake, storage, and utilization ultimately results from the interplay between the Fur regulon, several other transcription factors, the FtsH3 protease, and the sRNA IsaR1.

Evaluation and comparison of multi-omics data integration methods for cancer subtyping.

Computational integrative analysis has become a significant approach in the data-driven exploration of biological problems. Many integration methods for cancer subtyping have been proposed, but evaluating these methods has become a complicated problem due to the lack of gold standards. Moreover, questions of practical importance remain to be addressed regarding the impact of selecting appropriate data types and combinations on the performance of integrative studies. Here, we constructed three classes of benchmarking datasets of nine cancers in TCGA by considering all the eleven combinations of four multi-omics data types. Using these datasets, we conducted a comprehensive evaluation of ten representative integration methods for cancer subtyping in terms of accuracy measured by combining both clustering accuracy and clinical significance, robustness, and computational efficiency. We subsequently investigated the influence of different omics data on cancer subtyping and the effectiveness of their combinations. Refuting the widely held intuition that incorporating more types of omics data always produces better results, our analyses showed that there are situations where integrating more omics data negatively impacts the performance of integration methods. Our analyses also suggested several effective combinations for most cancers under our studies, which may be of particular interest to researchers in omics data analysis.

Well-Being of Teachers: The Role of Efficacy of Teachers and Academic Optimism.

In previous decades, the well-being of teachers has been at the center of the attention of researchers in several practical investigations on various subjects such as language learning. The objective of this review is to clarify this construct and add new information on the predominance of the well-being of teachers and organize factors impacting it. Nevertheless, among factors influencing the levels of well-being, the focus of this review is on two constructs, namely, optimism "as a new concept in positive psychology," societal theory, and collective school assets in education, and efficacy of teachers as an individual source in teachers. This review tries to focus on the eminent role of teacher efficacy and optimism in the process of education and also clarify their consequences on the well-being of teachers. In a nutshell, this review can provide implications for language teaching participants in the academic context.

CEPICS: A Comparison and Evaluation Platform for Integration Methods in Cancer Subtyping.

Cancer subtypes can improve our understanding of cancer, and suggest more precise treatment for patients. Multi-omics molecular data can characterize cancers at different levels. Up to now, many computational methods that integrate multi-omics data for cancer subtyping have been proposed. However, there are no consistent criteria to evaluate the integration methods due to the lack of gold standards (e.g., the number of subtypes in a specific cancer). Since comprehensive evaluation and comparison between different methods serves as a useful tool or guideline for users to select an optimal method for their own purpose, we develop a scalable platform, CEPICS, for comprehensively evaluating and comparing multi-omics data integration methods in cancer subtyping. Given a user-specified maximum number of subtypes, k-max, CEPICS provides (1) cancer subtyping results using up to five built-in state-of-the-art integration methods under the number of subtypes from two to k-max, (2) a report including the evaluation of each user-selected method and comparisons across them using clustering performance metrics and clinical survival analysis, and (3) an overall analysis of subtyping results by different methods representing a robust cancer subtype prediction for samples. Furthermore, users can upload subtyping results of their own methods to compare with the built-in methods. CEPICS is implemented as an R package and is freely available at https://github.com/GaoLabXDU/CEPICS.

The primary transcriptome of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

BACKGROUND: Cyanobacteria have shown promising potential for the production of various biofuels and chemical feedstocks. Synechococcus elongatus UTEX 2973 is a fast-growing strain with pronounced tolerance to high temperatures and illumination. Hence, this strain appears to be ideal for the development of photosynthetic biotechnology. However, molecular insights on how this strain can rapidly accumulate biomass and carbohydrates under high-light and high-temperature conditions are lacking. RESULTS: Differential RNA-Sequencing (dRNA-Seq) enabled the genome-wide identification of 4808 transcription start sites (TSSs) in S. elongatus UTEX 2973 using a background reduction algorithm. High light promoted the transcription of genes associated with central metabolic pathways, whereas the highly induced small RNA (sRNA) PsrR1 likely contributed to the repression of phycobilisome genes and the accelerated glycogen accumulation rates measured under this condition. Darkness caused transcriptome remodeling with a decline in the expression of genes for carbon fixation and other major metabolic pathways and an increase in the expression of genes for glycogen catabolism and Calvin cycle inhibitor CP12. Two of the identified TSSs drive the transcription of highly abundant sRNAs in darkness. One of them is widely conserved throughout the cyanobacterial phylum. Its gene is fused to a protein-coding gene in some species, illustrating the evolutionary origin of sRNAs from an mRNA 3'-end. CONCLUSIONS: Our comprehensive set of genome-wide mapped TSSs, sRNAs and promoter activities will be valuable for projects requiring precise information about the control of transcription aimed at metabolic engineering and the elucidation of stress acclimation mechanisms in this promising strain.

A Specific Single Nucleotide Polymorphism in the ATP Synthase Gene Significantly Improves Environmental Stress Tolerance of Synechococcus elongatus PCC 7942.

In response to a broad range of habitats and environmental stresses, cyanobacteria have evolved various effective acclimation strategies, which will be helpful for improving the stress tolerances of photosynthetic organisms, including higher plants. Synechococcus elongatus UTEX 2973 and PCC 7942 possess genomes that are 99.8% identical but exhibit significant differences in cell growth and stress tolerance. In this study, we found that a single amino acid substitution at FoF1 ATP synthase subunit α (AtpA), C252Y, is the primary contributor to the improved stress tolerance of S. elongatus UTEX 2973. Site-saturation mutagenesis experiments showed that point mutations of cysteine 252 to any of the four conjugated amino acids could significantly improve the stress tolerance of S. elongatus PCC 7942. We further confirmed that the C252Y mutation increases AtpA protein levels, intracellular ATP synthase activity, intracellular ATP abundance, transcription of psbA genes (especially psbA2), photosystem II activity, and glycogen accumulation in S. elongatus PCC 7942. This work highlights the importance of AtpA in improving the stress tolerance of cyanobacteria and provides insight into how cyanobacteria evolve via point mutations in the face of environmental selection pressures.IMPORTANCE Two closely related Synechococcus strains showed significantly different tolerances to high light and high temperature but limited genomic differences, providing us opportunities to identify key genes responsible for stress acclimation by a gene complementation approach. In this study, we confirmed that a single point mutation in the α subunit of FoF1 ATP synthase (AtpA) contributes mainly to the improved stress tolerance of Synechococcus elongatus UTEX 2973. The point mutation of AtpA, the important ATP-generating complex of photosynthesis, increases AtpA protein levels, intracellular ATP synthase activity, and ATP concentrations under heat stress, as well as photosystem II activity. This work proves the importance of ATP synthase in cyanobacterial stress acclimation and provides a good target for future improvement of cyanobacterial stress tolerance by metabolic engineering.

Enzymatic Activity Assay for Invertase in Synechocystis Cells.

Invertase can catalyze the hydrolysis of sucrose, and is widely distributed in cells of cyanobacteria and plants. Being responsible for the first step for sucrose metabolism, invertase plays important physiological roles and its enzymatic activity is frequently needed to be determined. All the methods for determination of the invertase activity are dependent on detection of the glucose product generated by the invertase. Here we describe an ion chromatography based protocol of our laboratory for determination of cyanobacterial intracellular invertase activity.

Determination of Intracellular Osmolytes in Cyanobacterial Cells.

Most of the cyanobacteria accumulate osmolytes including sucrose, glucosylglycerol, in their cells in response to salt stress. Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular sucrose and glucosylglycerol. We have confirmed this protocol was applicable to at least four kinds of cyanobacteria, filamentous cyanobacterium Anabaena sp. PCC 7120, unicellular cyanobacterium Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942 and halotolerant unicellular cyanobacterium Synechococcus sp. PCC 7002.

The Response Regulator Slr1588 Regulates spsA But Is Not Crucial for Salt Acclimation of Synechocystis sp. PCC 6803.

Cyanobacterial sucrose biosynthesis is stimulated under salt stress, which could be used for biotechnological sugar production. It has been shown that the response regulator Slr1588 negatively regulates the spsA gene encoding sucrose-phosphate synthase and mutation of the slr1588 gene also affected the salt tolerance of Synechocystis (Chen et al., 2014). The latter finding is contrary to earlier observations (Hagemann et al., 1997b). Here, we observed that ectopic expression of slr1588 did not restore the salt tolerance of the slr1588 mutant, making the essential function of this response regulator for salt tolerance questionable. Subsequent experiments showed that deletion of the entire coding sequence of slr1588 compromised the expression of the downstream situated ggpP gene, which encodes glucosylglycerol-phosphate phosphatase for synthesis of the primary osmolyte glucosylglycerol. Mutation of slr1588 by deleting the N-terminal part of this protein (Δslr1588-F976) did not affect ggpP expression, glucosylglycerol accumulation as well as salt tolerance, while the mutation of ggpP resulted in the previously reported salt-sensitive phenotype. In the Δslr1588-F976 mutant spsA was up-regulated but sucrose content was lowered due to increased invertase activity. Our results reveal that Slr1588 is acting as a repressor for spsA as previously suggested but it is not crucial for the overall salt acclimation of Synechocystis.

Slr1670 from Synechocystis sp. PCC 6803 Is Required for the Re-assimilation of the Osmolyte Glucosylglycerol.

When subjected to mild salt stress, the cyanobacterium Synechocystis sp. PCC 6803 produces small amounts of glycerol through an as of yet unidentified pathway. Here, we show that this glycerol is a degradation product of the main osmolyte of this organism, glucosylglycerol (GG). Inactivation of ggpS, encoding the first step of GG-synthesis, abolished de novo synthesis of glycerol, while the ability to hydrolyze exogenously supplied glucoslylglycerol was unimpaired. Inactivation of glpK, encoding glycerol kinase, had no effect on glycerol synthesis. Inactivation of slr1670, encoding a GHL5-type putative glycoside hydrolase, abolished de novo synthesis of glycerol, as well as hydrolysis of GG, and led to increased intracellular concentrations of this osmolyte. Slr1670 therefore presumably displays GG hydrolase activity. A gene homologous to the one encoded by slr1670 occurs in a wide range of cyanobacteria, proteobacteria, and archaea. In cyanobacteria, it co-occurs with genes involved in GG-synthesis.

The potential of Synechococcus elongatus UTEX 2973 for sugar feedstock production.

It is important to obtain abundant sugar feedstocks economically and sustainably for bio-fermentation industry, especially for producing cheap biofuels and biochemicals. Besides plant biomass, photosynthetic cyanobacteria have also been considered to be potential microbe candidates for sustainable production of carbohydrate feedstocks. As the fastest growing cyanobacterium reported so far, Synechococcus elongatus UTEX 2973 (Syn2973) might have huge potential for bioproduction. In this study, we explored the potentials of this strain as photo-bioreactors for sucrose and glycogen production. Under nitrogen-replete condition, Syn2973 could accumulate glycogen with a rate of 0.75 g L(-1) day(-1) at the exponential phase and reach a glycogen content as high as 51 % of the dry cell weight (DCW) at the stationary phase. By introducing a sucrose transporter CscB, Syn2973 was endowed with an ability to secrete over 94 % sucrose out of cells under salt stress condition. The highest extracellular sucrose productivity reached 35.5 mg L(-1) h(-1) for the Syn2973 strain expressing cscB, which contained the similar amounts of intracellular glycogen with the wild type. Potassium chloride was firstly proved to induce sucrose accumulation as well as sodium chloride in Syn2973. By semi-continuous culturing, 8.7 g L(-1) sucrose was produced by the cscB-expressing strain of Syn2973 in 21 days. These results support that Syn2973 is a promising candidate with great potential for production of sugars.

Study on active ingredient and mechanism in preventing vascular dementia of Tianzhusan coming from Tujia medicine.

To make clear of the absorbed components of Tianzhusan (TZS) and its possible mechanism in preventing vascular dementia (VD), the rats' models of VD were prepared by a permanent ligation of the bilateral common carotid arteries. After 60 days, rats were administrated with TZS for 0.1 g x kg(-1), and the volume is 0.02 mL x g(-1). After 3 days, the medicated serum was prepared and detected by UPLC, and then we predicted the possible chemical structure of the absorbed components of TZS. According to the absorbed components, the potential targets of TZS were found by ligand profiling of Discovery Studio 3.5. All of these target genes were submitted to DAVID onine for gene set enrichment analysis (GSEA). The 5 absorbed components of TZS have been predicted, and four of them have been identified as parishin B, parishin C, parishin, pennogenin-3-O-alpha-L-rhamnopyranosy-(1-->2)-beta-D-glucoside. Through reverse finding targets, we got 861 pharmacophore models and 9 pathways from KEGG, BIOCARTA after document verification. These results showed that the efficacy mechanism of TZS on VD perhaps were be related with these absorbed components and pathways. If the traditional herbs could be proved effective by efficacy tests, the serum pharmacochemistry, computer-aided drug design, system biology and other technologies can be used in the next experiments, which will be beneficial to fast discovery of material basis and mechanisms of traditional medicine coming form ethnic minorities.

A versatile strategy toward binary three-dimensional architectures based on engineering graphene aerogels with porous carbon fabrics for supercapacitors.

Graphene-based supercapacitors and related flexible devices have attracted great attention because of the increasing demands in the energy storage. As promising three-dimensional (3D) nanostructures in the supercapacitor electrodes, graphene-based aerogels have been paid dramatic attention recently, and numerous methods have been developed for enhancing their performance in energy storage. In this study, an exclusive strategy is presented toward directly in situ growing reduced graphene oxide (RGO) aerogels inside the 3D porous carbon fabrics for engineering the interfaces of the resulting binary 3D architectures. Such unique architectures have shown various advantages in the improvements of the nanostructures and chemical compositions, allowing them to possess much enhanced electrochemical properties (391, 229, and 195 F g(-1) at current densities of 0.1, 1, and 5 A g(-1), respectively) with excellent cycling stability in comparison with the neat RGO aerogels. The results of the performance in the flexible all-solid-state supercapacitors along with discussion on the related mechanisms in the electrochemical properties indicate the remaining issues and associated opportunities in the development of advanced energy storage devices. This strategy is relatively facile, versatile, and tunable, which highlights a unique platform for engineering various 3D porous structures in many fields.