Bacteriophage protein Gp46 is a cross-species inhibitor of nucleoid-associated HU proteins.

The architectural protein histone-like protein from Escherichia coli strain U93 (HU) is the most abundant bacterial DNA binding protein and highly conserved among bacteria and Apicomplexan parasites. It not only binds to double-stranded DNA (dsDNA) to maintain DNA stability but also, interacts with RNAs to regulate transcription and translation. Importantly, HU is essential to cell viability for many bacteria; hence, it is an important antibiotic target. Here, we report that Gp46 from bacteriophage SPO1 of Bacillus subtilis is an HU inhibitor whose expression prevents nucleoid segregation and causes filamentous morphology and growth defects in bacteria. We determined the solution structure of Gp46 and revealed a striking negatively charged surface. An NMR-derived structural model for the Gp46-HU complex shows that Gp46 occupies the DNA binding motif of the HU and therefore, occludes DNA binding, revealing a distinct strategy for HU inhibition. We identified the key residues responsible for the interaction that are conserved among HUs of bacteria and Apicomplexans, including clinically significant Mycobacterium tuberculosis, Acinetobacter baumannii, and Plasmodium falciparum, and confirm that Gp46 can also interact with these HUs. Our findings provide detailed insight into a mode of HU inhibition that provides a useful foundation for the development of antibacteria and antimalaria drugs.

Accumulation mechanism of metabolite markers identified by machine learning between Qingyuan and Xiushui counties in Polygonatum cyrtonema Hua.

Polygonatum cyrtonema Hua is a traditional Chinese medicinal plant acclaimed for its therapeutic potential in diabetes and various chronic diseases. Its rhizomes are the main functional parts rich in secondary metabolites, such as flavonoids and saponins. But their quality varies by region, posing challenges for industrial and medicinal application of P. cyrtonema. In this study, 482 metabolites were identified in P. cyrtonema rhizome from Qingyuan and Xiushui counties. Cluster analysis showed that samples between these two regions had distinct secondary metabolite profiles. Machine learning methods, specifically support vector machine-recursive feature elimination and random forest, were utilized to further identify metabolite markers including flavonoids, phenolic acids, and lignans. Comparative transcriptomics and weighted gene co-expression analysis were performed to uncover potential candidate genes including CHI, UGT1, and PcOMT10/11/12/13 associated with these compounds. Functional assays using tobacco transient expression system revealed that PcOMT10/11/12/13 indeed impacted metabolic fluxes of the phenylpropanoid pathway and phenylpropanoid-related metabolites such as chrysoeriol-6,8-di-C-glucoside, syringaresinol-4'-O-glucopyranosid, and 1-O-Sinapoyl-D-glucose. These findings identified metabolite markers between these two regions and provided valuable genetic insights for engineering the biosynthesis of these compounds.

Multi-scale nested UNet with transformer for colorectal polyp segmentation.

BACKGROUND: Polyp detection and localization are essential tasks for colonoscopy. U-shape network based convolutional neural networks have achieved remarkable segmentation performance for biomedical images, but lack of long-range dependencies modeling limits their receptive fields. PURPOSE: Our goal was to develop and test a novel architecture for polyp segmentation, which takes advantage of learning local information with long-range dependencies modeling. METHODS: A novel architecture combining with multi-scale nested UNet structure integrated transformer for polyp segmentation was developed. The proposed network takes advantage of both CNN and transformer to extract distinct feature information. The transformer layer is embedded between the encoder and decoder of a U-shape net to learn explicit global context and long-range semantic information. To address the challenging of variant polyp sizes, a MSFF unit was proposed to fuse features with multiple resolution. RESULTS: Four public datasets and one in-house dataset were used to train and test the model performance. Ablation study was also conducted to verify each component of the model. For dataset Kvasir-SEG and CVC-ClinicDB, the proposed model achieved mean dice score of 0.942 and 0.950 respectively, which were more accurate than the other methods. To show the generalization of different methods, we processed two cross dataset validations, the proposed model achieved the highest mean dice score. The results demonstrate that the proposed network has powerful learning and generalization capability, significantly improving segmentation accuracy and outperforming state-of-the-art methods. CONCLUSIONS: The proposed model produced more accurate polyp segmentation than current methods on four different public and one in-house datasets. Its capability of polyps segmentation in different sizes shows the potential clinical application.

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins.

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Missing puzzle pieces of time-restricted-eating (TRE) as a long-term weight-loss strategy in overweight and obese people? A systematic review and meta-analysis of randomized controlled trials.

The efficacy of using time restricted eating (TRE) for weight management and to mitigate metabolic disorders in overweight and obese people remains debatable. This meta-analysis quantified the impact of TRE on weight loss and metabolic health in overweight and obese people. The pooled results were subjected to a random-effects modeling using Hartung-Knapp-Sidik-Jonkman (HKSJ) method. Additionally, subgroup analysis was conducted based on study types, randomized controlled trials (RCTs) vs. non-randomized studies of interventions (NRSIs). Pooled results showed that subjects on TRE regimen (> 4 weeks) achieved a significant weight loss in comparison with unrestricted time regimen (weighted mean difference: -2.32%; 95% CI: -3.50, -1.14%; p < 0.01); however, weight loss was mainly attributed to the loss of lean mass rather than fat mass. The magnitude of weight loss was inversely correlated with daily fasting duration in RCTs. TRE significantly decreased the diastolic blood pressure and fasting insulin. An increase of low-density lipoprotein cholesterol (LDL-C) was observed in the TRE group. Favorable effect of TRE was observed on glucose metabolism but not on lipid profiles independent of weight loss. Hence TRE shall be administered with caution to overweight and obese people who have comorbidities such as dyslipidemia and sarcopenia.Supplemental data for this article is available online at https://doi.org/10.1080/10408398.2021.1974335.

Perturbing BEAMs: EEG adversarial attack to deep learning models for epilepsy diagnosing.

Deep learning models have been widely used in electroencephalogram (EEG) analysis and obtained excellent performance. But the adversarial attack and defense for them should be thoroughly studied before putting them into safety-sensitive use. This work exposes an important safety issue in deep-learning-based brain disease diagnostic systems by examining the vulnerability of deep learning models for diagnosing epilepsy with brain electrical activity mappings (BEAMs) to white-box attacks. It proposes two methods, Gradient Perturbations of BEAMs (GPBEAM), and Gradient Perturbations of BEAMs with Differential Evolution (GPBEAM-DE), which generate EEG adversarial samples, for the first time by perturbing BEAMs densely and sparsely respectively, and find that these BEAMs-based adversarial samples can easily mislead deep learning models. The experiments use the EEG data from CHB-MIT dataset and two types of victim models each of which has four different deep neural network (DNN) architectures. It is shown that: (1) these BEAM-based adversarial samples produced by the proposed methods in this paper are aggressive to BEAM-related victim models which use BEAMs as the input to internal DNN architectures, but unaggressive to EEG-related victim models which have raw EEG as the input to internal DNN architectures, with the top success rate of attacking BEAM-related models up to 0.8 while the top success rate of attacking EEG-related models only 0.01; (2) GPBEAM-DE outperforms GPBEAM when they are attacking the same victim model under a same distortion constraint, with the top attack success rate 0.8 for the former and 0.59 for the latter; (3) a simple modification to the GPBEAM/GPBEAM-DE will make it have aggressiveness to both BEAMs-related and EEG-related models (with top attack success rate 0.8 and 0.64), and this capacity enhancement is done without any cost of distortion increment. The goal of this study is not to attack any of EEG medical diagnostic systems, but to raise concerns about the safety of deep learning models and hope to lead to a safer design.

Study on the ozonation degradation of methylene blue enhanced by microchannel and ultrasound.

Azo dye-containing wastewater poses serious risks of environmental pollution because it is generally biologically toxic and resistant to conventional wastewater treatment methods. A novel degradation system integrating ozone, microchannel, and ultrasound was designed to effectively degrade azo dye-contaminated wastewater. The effects of discharge voltage of dielectric barrier discharge (DBD) reactor, liquid flow rate, microchannel width, ultrasonic power, initial pH, and reaction temperature on methylene blue (MB) decolorization were studied. A maximum MB decolorization efficiency of 92.7% was obtained in the ozone/microchannel/ultrasound (O3/MC/US) system with 14 min of treatment. In addition, the 14-min decolorization efficiency and TOC removal efficiency obtained in O3/MC/US system were increased by 12.6 and 6.5%, respectively, compared to those obtained in the pure O3 system. Based on the results of scavenging experiments, the combined effects of microchannel and ultrasound were proved to improve the contribution rate of hydroxyl radicals, thus improving the decolorization efficiency. The present work clearly illustrates that ozonation degradation can be effectively enhanced by microchannel and ultrasound, and also provides a feasible method for the treatment of organic wastewater.

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly.

Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium Synechocystis sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis.

Effectiveness of a novel traction device in endoscopic submucosal dissection for colorectal lesions.

BACKGROUND: Among all types of superficial gastrointestinal (GI) neoplasms, colorectal lesions are recognized as one of the most difficult locations to operate, due to the limited operation space, physiological bends, poor visualization of the submucosal dissection plane sheltered by colorectal crinkle wall, and the thin intestinal mucosa layer which is easy to perforation. The purpose of this prospective study is to evaluate the feasibility, efficacy, and safety of a novel endoscopic traction technique in assisting the endoscopic submucosal dissection (ESD) procedure in colorectal lesions. METHOD: A total of 117 patients with colonic lesions who underwent endoscopic treatment were enrolled between August 2020 and January 2021 at the endoscopic center of Beijing Chao-yang Hospital of Capital Medical University. Based on whether traction device was used during the operation, 60 and 57 patients were assigned to the conventional ESD group and clips and rubber band triangle traction-assisted ESD group (CRT-ESD, in which three clips and a rubber band were used to form an elastic triangular traction device), respectively. The total procedure time (TPT), submucosal dissection time (SDT), submucosal dissection speed (SDS), and rate of adverse events of the two groups were analyzed. RESULTS: After excluding patients who did not undergo treatment (conventional ESD, 1; CRT-ESD, 4), 112 patients were included in the study (conventional ESD, 59; CRT-ESD, 53). The baseline characteristics of the patients were well balanced between the two groups. The TPT (58.71 ± 26.22 min vs 33.58 ± 9.88 min, p < 0.001) and SDT (49.24 ± 23.75 min vs 26.34 ± 8.75 min, p < 0.001) were significantly different between the conventional ESD group and CRT-ESD group. The CRT-ESD group had significantly higher SDS than that of the traditional ESD group (0.54 ± 0.42 cm2/min vs 0.89 ± 0.40 cm2/min, p < 0.001). There were 4 (6.8%) cases of perforation in the traditional ESD group, and no perforation occurred in traction-assisted ESD. CONCLUSIONS: Compared with traditional ESD, CRT-ESD with clip and rubber band is both safer and more effective in the treatment of colorectal lesions.

Probing the electric field across thylakoid membranes in cyanobacteria.

In plants, algae, and some photosynthetic bacteria, the ElectroChromic Shift (ECS) of photosynthetic pigments, which senses the electric field across photosynthetic membranes, is widely used to quantify the activity of the photosynthetic chain. In cyanobacteria, ECS signals have never been used for physiological studies, although they can provide a unique tool to study the architecture and function of the respiratory and photosynthetic electron transfer chains, entangled in the thylakoid membranes. Here, we identified bona fide ECS signals, likely corresponding to carotenoid band shifts, in the model cyanobacteria Synechococcus elongatus PCC7942 and Synechocystis sp. PCC6803. These band shifts, most likely originating from pigments located in photosystem I, have highly similar spectra in the 2 species and can be best measured as the difference between the absorption changes at 500 to 505 nm and the ones at 480 to 485 nm. These signals respond linearly to the electric field and display the basic kinetic features of ECS as characterized in other organisms. We demonstrate that these probes are an ideal tool to study photosynthetic physiology in vivo, e.g., the fraction of PSI centers that are prebound by plastocyanin/cytochrome c6 in darkness (about 60% in both cyanobacteria, in our experiments), the conductivity of the thylakoid membrane (largely reflecting the activity of the ATP synthase), or the steady-state rates of the photosynthetic electron transport pathways.

The cAMP-Adenosine Feedback Loop Maintains the Suppressive Function of Regulatory T Cells.

Therapeutic manipulation of regulatory T cells (Tregs) has been regarded as a promising approach for the treatment of immune disorders. However, a better understanding of the immunomodulatory mechanisms of Tregs and new safe and effective methods to improve the therapeutic effects of Tregs are highly desired. In this study, we have identified the key roles of a cAMP-adenosine positive feedback loop in the immunomodulatory function of Tregs. Adult male C57BL/6J mice were used for an experimental autoimmune uveitis (EAU) model, Tregs, and uveitogenic T cells (UTs). In established EAU, induced Tregs (iTregs) administration alleviated the inflammatory response. In vitro, iTregs inhibited UTs proliferation and inflammatory cytokine production. Mechanistically, cAMP is partially responsible for iTreg-mediated inhibition on UTs. Importantly, intracellular cAMP regulates CD39 expression and CD39-dependent adenosine production in iTregs, and cAMP directly participates in iTreg-derived adenosine production by a CD39 signaling-independent extracellular cAMP-adenosine pathway. Moreover, extracellular adenosine increases the intracellular cAMP level in Tregs. More importantly, increasing the cAMP level in iTregs before transfer improves their therapeutic efficacy in established EAU. Notably, the cAMP-adenosine loop exists in both iTregs and naturally occurring Tregs. These findings provide new insights into the immunosuppressive mechanisms of Tregs and suggest a new strategy for improving the therapeutic efficacy of Tregs in established autoimmune disease.

Transcriptomics and Metabolomics Analyses Provide Novel Insights into Glucose-Induced Trophic Transition of the Marine Diatom Nitzschia laevis.

Diatoms have important ecological roles and are natural sources of bioactive compounds. Nitzschia laevis is a member of marine diatoms that accumulates high-value products including fucoxanthin and eicosapentaenoic acid (EPA). In this study, physiological data showed that comparing to autotrophic growth, mixotrophic cultivation with glucose supplementation led to a decrease of chlorophyll and fucoxanthin content in N. laevis, and an increase of biomass density and EPA yield. To further examine the metabolic barriers for fucoxanthin and EPA biosynthesis, comparative transcriptomic and metabolome analyses were conducted, with a focus on the genes related to carotenoids biosynthesis and fatty acid metabolism. The results indicated that phytoene desaturase (PDS) and zeta-carotene isomerase (ZISO) could be the rate-limiting enzymes in carotenoid biosynthesis. The transcription regulation of 3-ketoacyl-CoA synthase (KCS) and elongation of very long chain fatty acids protein (EVOVL) are important contributors associated with polyunsaturated fatty acids (PUFAs) accumulation. Furthermore, we also investigated the glucose-associated regulatory genes using weighted gene co-expression network analysis, and identified potential hub genes linked with cell cycle, carbohydrate metabolism, purine biosynthesis, and lipid metabolism. This study offers a high-quality transcriptome resource for N. laevis and provides a molecular framework for further metabolic engineering studies on fucoxanthin and EPA production.

Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein.

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

The Photosystem II Assembly Factor Ycf48 from the Cyanobacterium Synechocystis sp. PCC 6803 Is Lipidated Using an Atypical Lipobox Sequence.

Photochemical energy conversion during oxygenic photosynthesis is performed by membrane-embedded chlorophyll-binding protein complexes. The biogenesis and maintenance of these complexes requires auxiliary protein factors that optimize the assembly process and protect nascent complexes from photodamage. In cyanobacteria, several lipoproteins contribute to the biogenesis and function of the photosystem II (PSII) complex. They include CyanoP, CyanoQ, and Psb27, which are all attached to the lumenal side of PSII complexes. Here, we show that the lumenal Ycf48 assembly factor found in the cyanobacterium Synechocystis sp. PCC 6803 is also a lipoprotein. Detailed mass spectrometric analysis of the isolated protein supported by site-directed mutagenesis experiments indicates lipidation of the N-terminal C29 residue of Ycf48 and removal of three amino acids from the C-terminus. The lipobox sequence in Ycf48 contains a cysteine residue at the -3 position compared to Leu/Val/Ile residues found in the canonical lipobox sequence. The atypical Ycf48 lipobox sequence is present in most cyanobacteria but is absent in eukaryotes. A possible role for lipoproteins in the coordinated assembly of cyanobacterial PSII is discussed.

Fabrication of Plano-Concave Plastic Lens by Novel Injection Molding Using Carbide-Bonded Graphene-Coated Silica Molds.

Injection molding of plastic optical lenses prevails over many other techniques in both efficiency and cost, however polymer shrinkage during cooling, high level of uneven residual stresses and refractive index variations have limited its potential use for high precision lenses fabrication. In this research, we adopted a newly-developed strong graphene network to both plain and convex fused silica mold surfaces and proposed a novel injection molding of plano-concave lenses with graphene coated fused silica molds. The unique combination of the graphene coating and fused silica substrate maximize the mechanical properties of the mold and coating materials, namely high hardness, low surface friction, and high heat preservation effect during cooling since fused silica has low thermal conductivity. This advanced injection molding process was implemented in molding of plano-concave lenses resulting in reduced polymer shrinkage. In addition, internal residual stresses, and refractive index variations were also analyzed and discussed in detail. Meanwhile, as a comparison of conventional injection mold material, aluminum mold inserts with the same shape and size were also diamond machined and then employed to mold the same plano-concave lenses. Finally, a simulation model using Moldex3D was utilized to interpret stress distributions of both graphene and aluminum molds and then validated by experiments. The comparison between graphene and aluminum molds reveals that the novel injection molding with carbide-bonded graphene coated fused silica mold inserts is capable of molding high quality optical lenses with much less shrinkage and residual stresses, but more uniform refractive index distribution.

Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis.

Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and ß-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.

Application of an Ultrafine Shearing Method for the Extraction of C-Phycocyanin from Spirulina platensis.

Cell disruption is an important step during the extraction of C-phycocyanin from Spirulina platensis. An ultrafine shearing method is introduced and combined with soaking and ultrasonication to disrupt the cell walls of S. platensis efficiently and economically. Five kinds of cell disruption method, including soaking, ultrasonication, freezing-thawing, soaking-ultrafine shearing and soaking-ultrafine shearing-ultrasonication were applied to break the cell walls of S. platensis. The effectiveness of cell breaking was evaluated based on the yield of the C-phycocyanin. The results show that the maximum C-phycocyanin yield was 9.02%, achieved by the soaking-ultrafine shearing-ultrasonication method, followed by soaking (8.43%), soaking-ultrafine shearing (8.89%), freezing and thawing (8.34%), and soaking-ultrasonication (8.62%). The soaking-ultrafine shearing-ultrasonication method is a novel technique for breaking the cell walls of S. platensis for the extraction of C-phycocyanin.

Sequential Combination of Microwave- and Ultrasound-Assisted Extraction of Total Flavonoids from Osmanthus fragrans Lour. Flowers.

Microwave-assisted and ultrasound-assisted extraction assays were used to isolate total flavonoids (TF) from Osmanthus fragrans flowers. The effects of the solid-liquid ratio, ethanol concentration, microwave power, microwave extraction time, ultrasonic power and ultrasonic extraction time on the yield of TF were studied. A sequential combination of microwave- and ultrasound-assisted extraction (SC-MUAE) methods was developed, which was subsequently optimized by Box-Behnken design-response surface methodology (BBD-RSM). The interaction effects of the ethanol concentration (40-60%), microwave extraction time (5-7 min), ultrasonic extraction time (8-12 min) and ultrasonic power (210-430 W) on the yield of TF were investigated. The optimum operating parameters for the extraction of TF were determined to be as follows: ethanol concentration (48.15%), microwave extraction time (6.43 min), ultrasonic extraction time (10.09 min) and ultrasonic power (370.9 W). Under these conditions, the extraction yield of TF was 7.86 mg/g.

Sub-cellular location of FtsH proteases in the cyanobacterium Synechocystis sp. PCC 6803 suggests localised PSII repair zones in the thylakoid membranes.

In cyanobacteria and chloroplasts, exposure to HL damages the photosynthetic apparatus, especially the D1 subunit of Photosystem II. To avoid chronic photoinhibition, a PSII repair cycle operates to replace damaged PSII subunits with newly synthesised versions. To determine the sub-cellular location of this process, we examined the localisation of FtsH metalloproteases, some of which are directly involved in degrading damaged D1. We generated transformants of the cyanobacterium Synechocystis sp. PCC6803 expressing GFP-tagged versions of its four FtsH proteases. The ftsH2-gfp strain was functional for PSII repair under our conditions. Confocal microscopy shows that FtsH1 is mainly in the cytoplasmic membrane, while the remaining FtsH proteins are in patches either in the thylakoid or at the interface between the thylakoid and cytoplasmic membranes. HL exposure which increases the activity of the Photosystem II repair cycle led to no detectable changes in FtsH distribution, with the FtsH2 protease involved in D1 degradation retaining its patchy distribution in the thylakoid membrane. We discuss the possibility that the FtsH2-GFP patches represent Photosystem II 'repair zones' within the thylakoid membranes, and the possible advantages of such functionally specialised membrane zones. Anti-GFP affinity pull-downs provide the first indication of the composition of the putative repair zones.

CyanoP is Involved in the Early Steps of Photosystem II Assembly in the Cyanobacterium Synechocystis sp. PCC 6803.

Although the PSII complex is highly conserved in cyanobacteria and chloroplasts, the PsbU and PsbV subunits stabilizing the oxygen-evolving Mn4CaO5 cluster in cyanobacteria are absent in chloroplasts and have been replaced by the PsbP and PsbQ subunits. There is, however, a distant cyanobacterial homolog of PsbP, termed CyanoP, of unknown function. Here we show that CyanoP plays a role in the early stages of PSII biogenesis in Synechocystis sp. PCC 6803. CyanoP is present in the PSII reaction center assembly complex (RCII) lacking both the CP47 and CP43 modules and binds to the smaller D2 module. A small amount of larger PSII core complexes co-purifying with FLAG-tagged CyanoP indicates that CyanoP can accompany PSII on most of its assembly pathway. A role in biogenesis is supported by the accumulation of unassembled D1 precursor and impaired formation of RCII in a mutant lacking CyanoP. Interestingly, the pull-down preparations of CyanoP-FLAG from a strain lacking CP47 also contained PsbO, indicating engagement of this protein with PSII at a much earlier stage in assembly than previously assumed.