Stromal Side Research Articles

Transcriptomics revealed the key molecular mechanisms of ofloxacin-induced hormesis in Chlorella pyrenoidosa at environmentally relevant concentration

Emerging pollutants such as antibiotics have aroused great concern in recent years. However, the knowledge of low concentration-induced hormesis was not well understood. This study evaluated and quantified hormetic effects of ofloxacin on Chlorella pyrenoidosa. LogNormal model predicted the maximal non-effect concentration was 0.13 mg/L and 2.96 mg/L at 3 and 21 d, respectively. The sensitive alterations in chlorophyll fluorescence suggested PSII was the main target. Transcriptomics revealed ofloxacin inhibited genes related to photosynthetic system while the cyclic electron around PSI decreased the pH value in stroma side and stimulated photoprotection via up-regulating psbS. The stimulation in citrate cycle pathway met the urgent requirements of energy for DNA replication and repair. In addition, the negative feedback of G3P in glycolysis pathway inhibited Calvin cycle. The degradation products illustrated the occurrence of multiple detoxification mechanisms such as demethylation and ring-opening. The mobilization of cytochrome P450 generated the constant detoxication of ofloxacin while glutathione was consumptively involved in biological binding. This study provided new insights into the molecular mechanisms of antibiotic-induced hormesis in microalgae.

The stromal side of the cytochrome b6f complex regulates state transitions.

In oxygenic photosynthesis, state transitions distribute light energy between PSI and PSII. This regulation involves reduction of the plastoquinone pool, activation of the state transitions 7 (STT7) protein kinase by the cytochrome (cyt) b6f complex, and phosphorylation and migration of light harvesting complexes II (LHCII). In this study, we show that in Chlamydomonas reinhardtii, the C-terminus of the cyt b6 subunit PetB acts on phosphorylation of STT7 and state transitions. We used site-directed mutagenesis of the chloroplast petB gene to truncate (remove L215b6) or elongate (add G216b6) the cyt b6 subunit. Modified complexes are devoid of heme ci and degraded by FTSH protease, revealing that salt bridge formation between cyt b6 (PetB) and Subunit IV (PetD) is essential to the assembly of the complex. In double mutants where FTSH is inactivated, modified cyt b6f accumulated but the phosphorylation cascade was blocked. We also replaced the arginine interacting with heme ci propionate (R207Kb6). In this modified complex, heme ci is present but the kinetics of phosphorylation are slower. We show that highly phosphorylated forms of STT7 accumulated transiently after reduction of the PQ pool and represent the active forms of the protein kinase. The phosphorylation of the LHCII targets is favored at the expense of the protein kinase, and the migration of LHCII toward PSI is the limiting step for state transitions.

Bilateral Crosslinking with Glutaraldehyde and 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide: An Optimization Strategy for the Application of Decellularized Human Amniotic Membrane in Tissue Engineering

Introduction. The decellularized human amniotic membrane (dHAM) emerges as a viable 3D scaffold for organ repair and replacement using a tissue engineering strategy. Glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) can increase the biomechanical properties of dHAM. However, the crosslinking process is associated with biochemical changes and residual toxic materials, dampening the biocompatibility of the dHAM. From a histologic point of view, each side of the amniotic membrane is biologically different. While the dHAM basement membrane side is rich in growth factors, the stromal side of the dHAM contains more connective tissue matrix (e.g., collagen fibers) which supports its biomechanical properties. Biocompatibility and biomechanical properties are two important challenges in the field of materials science. In this study, for the first time, the stromal and basement membrane side are cross-linked with GTA and EDC, respectively, to optimize the biocompatibility of the treated dHAM while sparing the GTA-mediated biomechanical improvements. Methods. Crosslinking was carried out on dHAM in three groups: EDC, GTA and bilateral treatment with EDC&GTA. Mechanical resistance, degradability, and crosslinking measurements were performed on treated dHAM. The viability of mesenchymal stem cells (MSCs) on the scaffolds was evaluated by the MTT assay. The expression levels of surface markers and images of the MSCs were thoroughly studied. Results. The results obtained showed that bilateral treatment of dHAM with EDC and GTA increased mechanical resistance. Similarly, the evaluation of surface markers revealed that bilaterally treated dHAM sustains the stemness and viability of MSCs at a level equal to that achieved with EDC alone. The SEM images indicated that the MSCs maintained adhesion on EDC&GTA-cross-linked dHAM. Conclusion. The current study explores a pioneering treatment of dHAM, a material long recognized for its regenerative properties, in a novel context. This research delves into the utilization of dHAM cross-linked with EDC&GTA, demonstrating its optimized efficacy in tissue engineering. The enhanced crosslinking technique significantly alters the membrane’s properties, amplifying its durability and therapeutic potential. In this novel bilateral treatment strategy (EDC and GTA), improving mechanical properties by GTA on the stromal surface and maintaining the biocompatibility of EDC on the side of the basement membrane of dHAM had been attained together. By investigating the handling and impact of this cross-linked membrane, this study unveils a new approach in leveraging a well-known material through an innovative process, revolutionizing its application in wound care.

Histopathological and statistical study of corneal repair in rabbits. A new way to understand corneal recovery.

The study aims to evaluate corneal healing post amniotic membrane transplantation in controlled corneal defects, justifying its application in routine ophthalmology practice. The objective is to establish a reliable method for assessing the repair process. In three groups of six adult New Zealand rabbits, keratectomy and a monolayer transplant of dehydrated human amniotic membrane (AM) were conducted in the left eye (OS) with the right eye (OD) serving as the control eye. Clinical signs were assessed, and both eyes were enucleated at 1, 2, and 4 weeks for optical coherence tomography (OCT) measurements and histological analysis, collecting data from different epithelium, stroma, and limbus regions. This study was conducted using a formula that combines histologic data categorizing their presence and/or type as beneficial for corneal repair. No statistically significant differences were found between the experimental and control eyes regarding all clinical signs and OCT measurements. However, a linear model using histopathological results showed a period-implant mode interaction with statistical significance (p=0.010). The use of the single-layer amniotic membrane resulted in improved corneal recovery with the stromal side showing better performance in the first week and the epithelial side proving to be more effective than the stromal side in the long term. For the first time, a statistical formula employing histopathological data is introduced to determine corneal recovery, potentially offering a more accurate and reliable method compared with the observation of clinical signs and corneal measurements with OCT.

Amine-Rich Hydrogels for Molecular Nanoarchitectonics of Photosystem II and Inverse Opal TiO2 toward Solar Water Oxidation.

Solar water oxidation is a crucial process in light-driven reductive synthesis, providing electrons and protons for various chemical reductions. Despite advances in light-harvesting materials and cocatalysts, achieving high efficiency and stability remains challenging. In this study, we present a simple yet effective strategy for immobilizing natural photosystems (PS) made of abundant and inexpensive elements, using amine-rich polyethylenimine (PEI) hydrogels, to fabricate organic/inorganic hybrid photoanodes. Natural PS II extracted from spinach was successfully immobilized on inverse opal TiO2 photoanodes in the presence of PEI hydrogels, leading to greatly enhanced solar water oxidation activity. Photoelectrochemical (PEC) analyses reveal that PS II can be immobilized in specific orientations through electrostatic interactions between the positively charged amine groups of PEI and the negatively charged stromal side of PS II. This specific orientation ensures efficient photogenerated charge separation and suppresses undesired side reactions such as the production of reactive oxygen species. Our study provides an effective immobilization platform and sheds light on the potential utilization of PS II in PEC water oxidation.

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts.

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

Экспериментальное обоснование использования 1% раствора гидроксипропилметилцеллюлозы для защиты эндотелия заднего послойного трансплантата роговицы при его выкраивании низкоэнергетическим фемтосекундным лазером по инвертированной методике

Purpose. To evaluate experimentally endothelial cell loss of animal's cornea during inverted posterior lamellar corneal graft preparation technique by low-energy femtosecond laser with and without ophthalmic viscosurgical device (OVD) – 1% hydroxypropyl methylcellulose (HPMC) solution application. Material and methods. Posterior lamellar corneal grafts were created using a femtosecond laser (FSL). In the control group (n=16), a few drops of corneal storage solution were applied to the endothelium just before applanation, in the experimental group (n=16) – 1% HPMС. The quality of the applanation were monitored using an integrated in laser OCT system. The endothelial viability was determined using live/dead cells assay and the grafts were examined on a confocal laser scanning microscope. The obtained images were analyzed to count live and dead endothelial cells (EC). To assess the state of collagen fibers of the stromal side of the graft, samples from the control and experimental groups were examined using a scanning electron microscope (SEM). Results. According to the OCT, in all samples of both groups, a flat profile of the contact zone between the laser head and the endothelial monolayer was observed during applanation. The difference between the number of live and dead ECs was statistically significant between both groups. There were 2854 [2819; 2879] live cells/mm² in the control group, and 3477 [3426; 3719] live cells/mm² in the experimental group (p<0.001). Thus, the number of dead ECs in the control group was 710 [649; 728] cells/mm² . The number of dead ECs in the experimental group was 402 [366; 427] cells/mm² (p<0.001). When analyzing the images obtained on a SEM, the preservation of the architectonics of the graft collagen fibers with their single defibrations in both groups was observed. Conclusion. Application of 1% HPMC provides protection for endothelial cells from damage during posterior laser dissection using an FSL. In the study group, the number of endothelial cells was 9.92% higher than in the control group. The presence of the OVD layer in the interface between the head of the FSL and the EC did not affect the formation of an effective laser cut and did not reduce the quality of the stromal surface of the graft. The inverted technique FSL cut using a 1% solution of HPMC on the surface of the endothelium may be recommended for use in clinical practice. Key words: endothelium, keratocytes, corneal transplantation, keratoplasty, posterior lamellar keratoplasty, femtosecond laser, ophthalmic viscosurgical device, hydroxypropyl methylcellulose, confocal microscopy, scanning electron microscopy

Cassia Angustifolia Primed ASCs Accelerate Burn Wound Healing by Modulation of Inflammatory Response.

Thermal traumas impose a huge burden on healthcare systems. This merits the need for advanced but cost-effective remedies with clinical prospects. In this context, we prepared a regenerative 3D-construct comprising of Cassia angustifolia extract (SM) primed adipose-derived stem cells (ASCs) laden amniotic membrane for faster burn wound repair. ASCs were preconditioned with SM (30µg/ml for 24h), and subsequently exposed to in-vitro thermal injury (51°C,10min). In-vivo thermal injury was induced by placing pre-heated copper-disc (2cm diameter) on dorsum of the Wistar rats. ASCs (2.0 × 105) pre-treated with SM (SM-ASCs), cultured on stromal side of amniotic membrane (AM) were transplanted in rat heat-injury model. Non-transplanted heat-injured rats and non-heat-injured rats were kept as controls. The significantly upregulated expression of IGF1, SDF1A, TGFβ1, VEGF, GSS, GSR, IL4, BCL2 genes and downregulation of BAX, IL6, TNFα, and NFkB1 in SM-ASCs in in-vitro and in-vivo settings confirmed its potential in promoting cell-proliferation, migration, angiogenesis, antioxidant, cell-survival, anti-inflammatory, and wound healing activity. Moreover, SM-ASCs induced early wound closure, better architecture, normal epidermal thickness, orderly-arranged collagen fibers, and well-developed skin appendages in healed rat-skin transplanted with AM+SM-ASCs, additionally confirmed by increased expression of structural genes (Krt1, Krt8, Krt19, Desmin, Vimentin, α-Sma) in comparison to untreated-ASCs laden-AM transplanted in heat injured rats. SM priming effectively enabled ASCs to counter thermal injury by significantly enhancing cell survival and reducing inflammation upon transplantation. This study provides bases for development of effective combinational therapies (natural scaffold, medicine, and stem cells) with clinical prospects for treating burn wounds.

Chloroplast import motor subunits FtsHi1 and FtsHi2 are located on opposite sides of the inner envelope membrane

Protein import into chloroplasts is powered by ATP hydrolysis in the stroma. Establishing the identity and functional mechanism of the stromal ATPase motor that drives import is critical for understanding chloroplast biogenesis. Recently, a complex consisting of Ycf2, FtsHi1, FtsHi2, FtsHi4, FtsHi5, FtsH12, and malate dehydrogenase was shown to be important for chloroplast protein import, and it has been proposed to act as the motor driving protein translocation across the chloroplast envelope into the stroma. To gain further mechanistic understanding of how the motor functions, we performed membrane association and topology analyses on two of its subunits, FtsHi1 and FtsHi2. We isolated cDNA clones encoding FtsHi1 and FtsHi2 preproteins to perform in vitro import experiments in order to determine the exact size of each mature protein. We also generated antibodies against the C-termini of the proteins, i.e., where their ATPase domains reside. Protease treatments and alkaline and high-salt extractions of chloroplasts with imported and endogenous proteins revealed that FtsHi1 is an integral membrane protein with its C-terminal portion located in the intermembrane space of the envelope, not the stroma, whereas FtsHi2 is a soluble protein in the stroma. We further complemented an FtsHi1-knockout mutant with a C-terminally tagged FtsHi1 and obtained identical results for topological analyses. Our data indicate that the model of a single membrane-anchored pulling motor at the stromal side of the inner membrane needs to be revised and suggest that the Ycf2-FtsHi complex may have additional functions.

Structure of Chlorella ohadii Photosystem II Reveals Protective Mechanisms against Environmental Stress.

Green alga Chlorella ohadii is known for its ability to carry out photosynthesis under harsh conditions. Using cryogenic electron microscopy (cryoEM), we obtained a high-resolution structure of PSII at 2.72 Å. This structure revealed 64 subunits, which encompassed 386 chlorophylls, 86 carotenoids, four plastoquinones, and several structural lipids. At the luminal side of PSII, a unique subunit arrangement was observed to protect the oxygen-evolving complex. This arrangement involved PsbO (OEE1), PsbP (OEE2), PsbB, and PsbU (a homolog of plant OEE3). PsbU interacted with PsbO, PsbC, and PsbP, thereby stabilizing the shield of the oxygen-evolving complex. Significant changes were also observed at the stromal electron acceptor side. PsbY, identified as a transmembrane helix, was situated alongside PsbF and PsbE, which enclosed cytochrome b559. Supported by the adjacent C-terminal helix of Psb10, these four transmembrane helices formed a bundle that shielded cytochrome b559 from the surrounding solvent. Moreover, the bulk of Psb10 formed a protective cap, which safeguarded the quinone site and likely contributed to the stacking of PSII complexes. Based on our findings, we propose a protective mechanism that prevents QB (plastoquinone B) from becoming fully reduced. This mechanism offers insights into the regulation of electron transfer within PSII.

The Unique Light-Harvesting System of the Algal Phycobilisome: Structure, Assembly Components, and Functions.

The phycobilisome (PBS) is the major light-harvesting apparatus in cyanobacteria and red algae. It is a large multi-subunit protein complex of several megadaltons that is found on the stromal side of thylakoid membranes in orderly arrays. Chromophore lyases catalyse the thioether bond between apoproteins and phycobilins of PBSs. Depending on the species, composition, spatial assembly, and, especially, the functional tuning of different phycobiliproteins mediated by linker proteins, PBSs can absorb light between 450 and 650 nm, making them efficient and versatile light-harvesting systems. However, basic research and technological innovations are needed, not only to understand their role in photosynthesis but also to realise the potential applications of PBSs. Crucial components including phycobiliproteins, phycobilins, and lyases together make the PBS an efficient light-harvesting system, and these provide a scheme to explore the heterologous synthesis of PBS. Focusing on these topics, this review describes the essential components needed for PBS assembly, the functional basis of PBS photosynthesis, and the applications of phycobiliproteins. Moreover, key technical challenges for heterologous biosynthesis of phycobiliproteins in chassis cells are discussed.

The journey of preproteins across the chloroplast membrane systems.

The photosynthetic capacity of chloroplasts is vital for autotrophic growth in algae and plants. The origin of the chloroplast has been explained by the endosymbiotic theory that proposes the engulfment of a cyanobacterium by an ancestral eukaryotic cell followed by the transfer of many cyanobacterial genes to the host nucleus. As a result of the gene transfer, the now nuclear-encoded proteins acquired chloroplast targeting peptides (known as transit peptides; transit peptide) and are translated as preproteins in the cytosol. Transit peptides contain specific motifs and domains initially recognized by cytosolic factors followed by the chloroplast import components at the outer and inner envelope of the chloroplast membrane. Once the preprotein emerges on the stromal side of the chloroplast protein import machinery, the transit peptide is cleaved by stromal processing peptidase. In the case of thylakoid-localized proteins, cleavage of the transit peptides may expose a second targeting signal guiding the protein to the thylakoid lumen or allow insertion into the thylakoid membrane by internal sequence information. This review summarizes the common features of targeting sequences and describes their role in routing preproteins to and across the chloroplast envelope as well as the thylakoid membrane and lumen.

Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.

A Model of Using the Asymmetric Polydopamine Thin Film for Mimicking Epithelial Folding In Vitro

AbstractThe basement membrane (BM) is a biointeractive ultrathin network with distinct composition and organization of its epithelial and stromal sides, which render BMs with asymmetric biofunctions and mechanical properties. There are difficulties in the recapitulation of the highly hierarchical structure and function of BM. Here, the interfacial assembly method for the generation of BM mimics is applied. Dopamine is the starting material for the polymerization and assembly of polydopamine (PDA) into asymmetric materials. Compared to the PDA coating formed at the solid/liquid interface (≈20 nm), the PDA film formed at the air/liquid interface displays a thickness of ≈100 nm. Moreover, it possesses an asymmetric surface topography and an apparent Young's modulus of ≈1.0 MPa, which is structurally and mechanically similar to natural BMs. Of interest, the airside and the waterside of the PDA film exhibit differences in their adhesion affinity to the human skin keratinocytes. With stronger active mechanical processes between living cells and the waterside of PDA film, epithelial folding could be mimicked. Together, the PDA film is able to recapitulate the structural and mechanical complexity of natural BMs, indicating the prospective future of using PDA films for in vitro modeling cell‐BM interaction and tissue formation.

Photoelectrochemistry of a photosystem I – Ferredoxin construct on ITO electrodes

In this study, photobioelectrodes based on a ferredoxin-modified photosystem I (PSI-Fd) from Thermosynechococcus vestitus have been prepared and characterized regarding the direct electron transfer between PSI-Fd and the electrode. The modified PSI with the covalently linked ferredoxin (Fd) on its stromal side has been immobilized on indium-tin-oxide (ITO) electrodes with a 3-dimensional inverse-opal structure. Compared to native PSI, a lower photocurrent and a lower onset potential of the cathodic photocurrent have been observed. This can be mainly attributed to a different adsorption behavior of the PSI-Fd-construct onto the 3D ITO. However, the overall behavior is rather similar to PSI. First experiments have been performed for applying this PSI-Fd photobioelectrode for enzyme-driven NADPH generation. By coupling the electrode system with ferredoxin-NADP+-reductase (FNR), first hints for the usage of photoelectrons for biosynthesis have been collected by verifying NADPH generation.

Adipose-derived human mesenchymal stem cells seeded on denuded or stromal sides of the amniotic membrane improve angiogenesis and collagen remodeling and accelerate healing of the full-thickness wound

Several strategies have been proposed to enhance wound healing results. Along with other forms of wound dressing, the human amniotic membrane (HAM) has long been regarded as a biological wound dressing that decreases infection and enhances healing. This study investigates the feasibility and effectiveness of wound healing using decellularized HAM (dAM) and stromal HAM (sAM) in combination with adipose-derived human mesenchymal stem cells (AdMSCs). The dAM and sAM sides of HAM were employed as wound dressing scaffolds, and AdMSCs were seeded on top of either dAM or sAM. Sixty healthy Wistar rats were randomly divided into three groups: untreated wound, dAM/AdMSCs group, and sAM/AdMSCs group. The gene expression of VEGF and COL-I was measured in vitro. Wound healing was examined after wounding on days 3, 7, 14, and 21. The expression level of VEGF was significantly higher in sAM/AdMSCs than dAM/AdMSCs (P ≤ 0.05), but there was no significant difference in COL-I expression (P ≥ 0.05). In vivo research revealed that on day 14, wounds treated with sAM/AdMSCs had more vascularization than wounds treated with dAM/AdMSCs (P ≤ 0.01) and untreated wound groups on days 7 (P ≤ 0.05) and 14 (P ≤ 0.0001), respectively. On days 14 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), and 21 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), the collagen deposition in the wound bed was significantly thicker in the sAM/AdMSCs and dAM/AdMSCs groups compared to untreated wounds. The study demonstrated that the combination of sAM and AdMSCs promotes wound healing by enhancing angiogenesis and collagen remodeling.

Insights into the binding mechanism of ascorbic acid and violaxanthin with violaxanthin de-epoxidase (VDE) and chlorophycean violaxanthin de-epoxidase (CVDE) enzymes.

Photosynthetic organisms have evolved to work under low and high lights in photoprotection, acting as a scavenger of reactive oxygen species. The light-dependent xanthophyll cycle involved in this process is performed by a key enzyme (present in the thylakoid lumen), Violaxanthin De-Epoxidase (VDE), in the presence of violaxanthin (Vio) and ascorbic acid substrates. Phylogenetically, VDE is found to be connected with an ancestral enzyme Chlorophycean Violaxanthin De-Epoxidase (CVDE), present in the green algae on the stromal side of the thylakoid membrane. However, the structure and functions of CVDE were not known. In search of functional similarities involving this cycle, the structure, binding conformation, stability, and interaction mechanism of CVDE are explored with the two substrates compared to VDE. The structure of CVDE was determined by homology modeling and validated. In silico docking (of first-principles optimized substrates) revealed it has a larger catalytic domain than VDE. A thorough analysis of the binding affinity and stability of four enzyme-substrate complexes is performed by computing free energies and their decomposition, the root-mean-square deviation (RMSD) and fluctuation (RMSF), the radius of gyration, salt bridge, and hydrogen bonding interactions in molecular dynamics. Based on these, violaxanthin interacts with CVDE to a similar extent as that of VDE. Hence, its role is expected to be the same for both enzymes. On the contrary, ascorbic acid has a weaker interaction with CVDE than VDE. Given these interactions drive epoxidation or de-epoxidation in the xanthophyll cycle, it immediately discerns that either ascorbic acid does not participate in de-epoxidation or a different cofactor is necessary as CVDE has a weaker interaction with ascorbic acid than VDE.

Femtosecond laser deep lamellar keratoplasty.

We aimed to develop a novel and effective technique for creating a smooth deep lamellar dissection of the cornea using a femtosecond (FS) laser for deep anterior lamellar keratoplasty (DALK), we conducted a retrospective eye bank study. Thirteen fresh human corneas were mounted on an artificial anterior chamber, and deep lamellar cuts were made with a 500-kHz VisuMax FS laser at a level of 50-80 μm anterior to the Descemet's membrane (DM). A posterior diameter of 8 mm with a side cut angle of 110° was used for the anterior penetrating side cut. The anterior lamellar tissue was bluntly dissected. The residual posterior stromal beds and side cuts were examined with microscopy and intraoperative optical coherence tomography (OCT) and post-cut endothelial cell evaluations. All corneas revealed a smooth residual posterior stromal bed without any visible irregularities or ridges by microscopy and OCT imaging. Six corneas were suitable for post-cut endothelial cell evaluation 2 days after laser cut, with no significant endothelial cell loss post-laser and blunt dissection of the posterior stroma. FS laser deep lamellar keratoplasty utilizing an ultrafast laser to produce a smooth deep stromal dissection followed by blunt dissection and removal of the anterior stromal tissue yields a consistent and smooth residual stromal bed. The creation of a smooth lamellar dissection in the deep posterior cornea may result in more consistent DALK without the need for air bubble or manual baring of DM that has the risk for DM perforation.

High efficient cyclic electron flow and functional supercomplexes in Chlamydomonas cells

A very high rate for cyclic electron flow (CEF) around PSI (~180 s−1 or 210 s−1 in minimum medium or in the presence of a carbon source respectively) is measured in the presence of methyl viologen (MV) in intact cells of Chlamydomonas reinhardtii under anaerobic conditions. The observation of an efficient CEF in the presence of methyl viologen is in agreement with the previous results reports of Asada et al. in broken chloroplasts (Plant Cell Physiol. 31(4) (1990) 557–564). From the analysis of the P700 and PC absorbance changes, we propose that a confinement between 2 PC molecules, 1 PSI and 1 cytb6f corresponding to a functional supercomplex is responsible for these high rates of CEF. Supercomplex formation is also observed in the absence of methyl viologen, but with lower maximal CEF rate (about 100 s−1) suggesting that this compound facilitates the mediation of electron transfer from PSI acceptors to the stromal side of cytb6f. Further analysis of CEF in mutants of Chlamydomonas defective in state transitions shows the requirement of a kinase-driven transition to state 2 to establish this functional supercomplex configuration. However, a movement of the LHCII antennae is not involved in this process. We discuss the possible involvement of auxiliary proteins, among which is a small cytb6f-associated polypeptide, the PETO protein, which is one of the targets of the STT7 kinase.