A Ubiquitin-Based Module Directing Protein-Protein Interactions in Chloroplasts.

A promising approach for the genetic engineering of multiprotein complexes in living cells involves designing and reconstructing the interaction between two proteins that lack native affinity. Thylakoid-embedded multiprotein complexes execute the light reaction of plant photosynthesis, but their engineering remains challenging, likely due to difficulties in accurately targeting heterologous membrane-bound proteins to various sub-compartments of thylakoids. In this study, we developed a ubiquitin-based module (Nub-Cub) capable of directing interactions in vivo between two chloroplast proteins lacking native affinities. We applied this module to genetically modify thylakoid multiprotein complexes. We demonstrated the functionality of the Nub-Cub module in the model organism Arabidopsis thaliana. Employing this system, we successfully modified the Photosystem II (PSII) complex by ectopically attaching an extrinsic subunit of PSII, PsbTn1, to CP26-a component of the antenna system of PSII. Surprisingly, this mandatory interaction between CP26 and PsbTn1 in plants impairs the efficiency of electron transport in PSII and unexpectedly results in noticeable defects in leaf development. Our study not only offers a general strategy to modify multiprotein complexes embedded in thylakoid membranes but it also sheds light on the possible interplay between two proteins without native interaction.

Sustained Vision Recovery by OSK Gene Therapy in a Mouse Model of Glaucoma.

Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases.

Evidence for Exciton Crystals in a 2D Semiconductor Heterotrilayer.

Two-dimensional (2D) transition metal dichalcogenides (TMDC) and their moiré interfaces have been demonstrated for correlated electron states, including Mott insulators and electron/hole crystals commensurate with moiré superlattices. Here we present spectroscopic evidence for ordered bosons─interlayer exciton crystals in a WSe2/MoSe2/WSe2 trilayer, where the enhanced Coulomb interactions over those in heterobilayers have been predicted to result in exciton ordering. Ordered interlayer excitons in the trilayer are characterized by negligible mobility and by sharper PL peaks persisting to an exciton density of nex ∼ 1012 cm-2, which is an order of magnitude higher than the corresponding limit in the heterobilayer. We present evidence for the predicted quadrupolar exciton crystal and its transitions to dipolar excitons either with increasing nex or by an applied electric field. These ordered interlayer excitons may serve as models for the exploration of quantum phase transitions and quantum coherent phenomena.

Efficient scavenging of reactive carbonyl species in chloroplasts is required for light acclimation and fitness of plants.

Reactive carbonyl species (RCS) derived from lipid peroxides can act as critical damage or signaling mediators downstream of reactive oxygen species by modifying target proteins. However, their biological effects and underlying mechanisms remain largely unknown in plants. Here, we have uncovered the mechanism by which the RCS 4-hydroxy-(E)-2-nonenal (HNE) participates in photosystem II (PSII) repair cycle of chloroplasts, a crucial process for maintaining PSII activity under high and changing light conditions. High Light Sensitive 1 (HLT1) is a potential NADPH-dependent reductase in chloroplasts. Deficiency of HLT1 had no impact on the growth of Arabidopsis plants under normal light conditions but increased sensitivity to high light, which resulted from a defective PSII repair cycle. In hlt1 plants, the accumulation of HNE-modified D1 subunit of PSII was observed, which did not affect D1 degradation but hampered the dimerization of repaired PSII monomers and reassembly of PSII supercomplexes on grana stacks. HLT1 is conserved in all photosynthetic organisms and has functions in overall growth and plant fitness in both Arabidopsis and rice under naturally challenging field conditions. Our work provides the mechanistic basis underlying RCS scavenging in light acclimation and suggests a potential strategy to improve plant productivity by manipulating RCS signaling in chloroplasts.

Microglia depletion exacerbates retinal ganglion cell loss in a mouse model of glaucoma.

To test whether depletion of microglia in the optic nerve head has a beneficial effect on retinal ganglion cell numbers and function, we depleted microglia by oral administration of the CSF1R antagonist PLX5622. Then, ocular hypertension was induced by unilateral injection of magnetic microbeads into the anterior chamber. Visual function was assessed with pattern electroretinography and measurement of the optomotor reflex. Retinal ganglion cell bodies and axons were counted and gene expression patterns in optic nerve head astrocytes were tested on freshly dissociated astrocytes. PLX5622 efficiently depleted microglia in the retina and the optic nerve head, but about 20% of microglia persisted in the myelinated optic nerve proper even after prolonged exposure to the drug. PLX5622 did not affect ganglion cell function by itself. Elevation of the IOP for four weeks led to the expected decrease in visual acuity and pattern ERG amplitude. Microglia ablation did not affect these parameters. Ganglion cell and axon numbers were counted histologically post mortem. Mice in the microglia depletion group showed a moderate but significantly greater loss of ganglion cells than the control group. At four weeks post microbead injection, gene expression patterns in optic nerve head astrocytes are consistent with an A2 (or neuroprotective) pattern. Microglia depletion blunted the up-regulation of A2 genes in astrocytes. In conclusion, microglia depletion is unlikely to protect retinal ganglion cells in early glaucoma.

NADP+ supply adjusts the synthesis of photosystem I in Arabidopsis chloroplasts.

In oxygenic photosynthesis, NADP+ acts as the final acceptor of the photosynthetic electron transport chain and receives electrons via the thylakoid membrane complex photosystem I (PSI) to synthesize NAPDH by the enzyme ferredoxin:NADP+ oxidoreductase. The NADP+/NADPH redox couple is essential for cellular metabolism and redox homeostasis. However, how the homeostasis of these two dinucleotides is integrated into chloroplast biogenesis remains largely unknown. Here, we demonstrate the important role of NADP+ supply for the biogenesis of PSI by examining the nad kinase 2 (nadk2) mutant in Arabidopsis (Arabidopsis thaliana), which demonstrates disrupted synthesis of NADP+ from NAD+ in chloroplasts. Although the nadk2 mutant is highly sensitive to light, the reaction center of photosystem II (PSII) is only mildly and likely only secondarily affected compared to the wild-type. Our studies revealed that the primary limitation of photosynthetic electron transport, even at low light intensities, occurs at PSI rather than at PSII in the nadk2 mutant. Remarkably, this primarily impairs the de novo synthesis of the two PSI core subunits PsaA and PsaB, leading to the deficiency of the PSI complex in the nadk2 mutant. This study reveals an unexpected molecular link between NADK activity and mRNA translation of psaA/B in chloroplasts that may mediate a feedback mechanism to adjust de novo biosynthesis of the PSI complex in response to a variable NADPH demand. This adjustment may be important to protect PSI from photoinhibition under conditions that favor acceptor side limitation.

Exosomal CagA from Helicobacter pylori aggravates intestinal epithelium barrier dysfunction in chronic colitis by facilitating Claudin-2 expression.

BACKGROUND: The chronic infection with Helicobacter pylori (H. pylori), especially cytotoxin-associated gene A-positive (CagA+) strains, has been associated with various extragastric disorders. Evaluating the potential impacts of virulence factor CagA on intestine may provide a better understanding of H. pylori pathogenesis such as colitis. The intestinal mucosal barrier is essential for maintaining its integrity and functions. However, how persistent CagA+ H. pylori colonization influences barrier disruption and thereby affects chronic colitis is not fully understood. RESULTS: Chronic colitis models of CagA+ H. pylori-colonized mice treated with 2% Dextran sulphate sodium (DSS) were established to assess the disease activity and pertinent expression of tight junction proteins closely related to mucosal integrity. The aggravating effect of CagA+ H. pylori infection on DSS-induced chronic colitis was confirmed in mouse models. In addition, augmented Claudin-2 expression was detected in CagA+ H. pylori infection conditions and selected for mechanistic analysis. Next, GES-1 human gastric epithelial cells were cultured with CagA+ H. pylori or a recombinant CagA protein, and exosomes isolated from conditioned media were then identified. We assessed the Claudin-2 levels after exposure to CagA+ exosomes, CagA- exosomes, and IFN-γ incubation, revealing that CagA+ H. pylori compromised the colonic mucosal barrier and facilitated IFN-γ-induced intestinal epithelial destruction through CagA-containing exosome-mediated mechanisms. Specifically, CagA upregulated Claudin-2 expression at the transcriptional level via a CDX2-dependent mechanism to slow the restoration of wounded mucosa in colitis in vitro. CONCLUSIONS: These data suggest that exosomes containing CagA facilitate CDX2-dependent Claudin-2 maintenance. The exosome-dependent mechanisms of CagA+ H. pylori infection are indispensable for damaging the mucosal barrier integrity in chronic colitis, which may provide a new idea for inflammatory bowel disease (IBD) treatment.

Detection of a novel PAX6 variant in a Chinese family with multiple ocular abnormalities.

BACKGROUND: Aniridia is a congenital, panocular disease that can affect the cornea, anterior chamber angle, iris, lens, retina and optic nerve. PAX6 loss-of-function variants are the most common cause of aniridia, and variants throughout the gene have been linked to a range of ophthalmic abnormalities. Furthermore, particular variants at a given site in PAX6 lead to distinct phenotypes. This study aimed to characterize genetic variants associated with congenital aniridia in a Chinese family. METHODS: The proband and family underwent ophthalmologic examinations. DNA was sampled from the peripheral blood of all 6 individuals, and whole-exome sequencing was performed. Sanger sequencing was used to verify the variant in this family members. RESULTS: A novel variant (c.114_119delinsAATTTCC: p.Pro39llefsTer17) in the PAX6 gene was identified in subjects II-1, III-1 and III-2, who exhibited complete aniridia and cataracts. The proband and the proband's brother also had glaucoma, high myopia, and foveal hypoplasia. CONCLUSIONS: We identified that a novel PAX6 frameshift heterozygous deletion variant is the predominant cause of aniridia in this Chinese family. TRIAL REGISTRATION: We did not perform any health-related interventions for the participants.

Helicobacter pylori Infection Acts as an Independent Risk Factor for Intracranial Atherosclerosis in Women Less Than 60 Years Old.

Background: Studies show inconsistent results regarding the relationship between Helicobacter pylori (H. pylori) infection and stroke. The present study assessed a potential association between H. pylori infection and an important risk factor for stroke, intracranial atherosclerosis. Methods: In total, 15,798 subjects with transcranial Doppler (TCD) and 13C-urea breath test (13C-UBT) were enrolled from March 2012 to March 2017. Intracranial atherosclerosis was further measured using intracranial carotid artery calcification (ICAC) on past or recent head CT, and 14,084 subjects were ultimately included in the study. Baseline demographics, atherosclerosis risk factors, and laboratory results were investigated. Since endothelial dysfunction is critical to the development of atherosclerosis, the role of H. pylori in migration, tube formation, and proliferation of human brain microvascular endothelial cells (HBMECs) was assessed in vitro. Results: The intracranial atherosclerosis group had a higher proportion of women and a greater rate of H. pylori infection than those without intracranial atherosclerosis. H. pylori infection was significantly more common in women with intracranial atherosclerosis than males. In addition, the incidence of intracranial atherosclerosis was significantly higher in women with H. pylori infection than uninfected women (53.8 vs. 46.4%, p < 0.001). In an adjusted model, H. pylori was shown to be an independent risk factor for intracranial atherosclerosis in women ≤ 60 years of age [odds ratio (OR) = 2.261, 95% CI = 1.839-2.780, p < 0.001]. Serum exosomes from patients with H. pylori infection had significantly reduced brain endothelial cell migration, tube formation, and proliferation in vitro. Conclusion: Helicobacter pylori infection may be an important independent risk factor for intracranial atherosclerosis in women ≤ 60 years of age.

Helicobacter pylori Infection Impairs Endothelial Function Through an Exosome-Mediated Mechanism.

Background Epidemiological studies have suggested an association between Helicobacter pylori (H pylori) infection and atherosclerosis through undefined mechanisms. Endothelial dysfunction is critical to the development of atherosclerosis and related cardiovascular diseases. The present study was designed to test the hypothesis that H pylori infection impaires endothelial function through exosome-mediated mechanisms. Methods and Results Young male and female patients (18-35 years old) with and without H pylori infection were recruited to minimize the chance of potential risk factors for endothelial dysfunction for the study. Endothelium-dependent flow-mediated vasodilatation of the brachial artery was evaluated in the patients and control subjects. Mouse infection models with CagA+H pylori from a gastric ulcer patient were created to determine if H pylori infection-induced endothelial dysfunction could be reproduced in animal models. H pylori infection significantly decreased endothelium-dependent flow-mediated vasodilatation in young patients and significantly attenuated acetylcholine-induced endothelium-dependent aortic relaxation without change in nitroglycerin-induced endothelium-independent vascular relaxation in mice. H pylori eradication significantly improved endothelium-dependent vasodilation in both patients and mice with H pylori infection. Exosomes from conditioned media of human gastric epithelial cells cultured with CagA+H pylori or serum exosomes from patients and mice with H pylori infection significantly decreased endothelial functions with decreased migration, tube formation, and proliferation in vitro. Inhibition of exosome secretion with GW4869 effectively preserved endothelial function in mice with H pylori infection. Conclusions H pylori infection impaired endothelial function in patients and mice through exosome-medicated mechanisms. The findings indicated that H pylori infection might be a novel risk factor for cardiovascular diseases.

Evolution and Expression Divergence of the CYP78A Subfamily Genes in Soybean.

Gene expression divergence is an important evolutionary driving force for the retention of duplicate genes. In this study, we identified three CYP78A subfamily genes in soybean, GmCYP78A70, GmCYP78A57 and GmCYP78A72, which experienced different duplication events. GmCYP78A70 was mainly expressed in leaf tissue and the vegetative phase, whereas GmCYP78A57 was mainly expressed in floral tissue and seed, i.e., the reproductive phase. Expression of GmCYP78A72 could be detected in all the tissues and phases mentioned above. The expression levels of GmCYP78A70 and GmCYP78A57 in different soybean cultivars showed positive correlations with leaf size and 100-seed weight, respectively. The population genetics analysis indicated that the three genes had experienced different selective pressures during domestication and improved breeding of soybean. Deciphering the function of this subfamily of genes may well prove useful to breeders for improving soybean's agronomic traits.

Effects of Helicobacter pylori on the expression levels of GATA-3 and connexin 32 and the GJIC function in gastric epithelial cells and their association by promoter analysis.

The present study aimed to explore the effects of Helicobacter pylori (H. pylori) infection on the expression of transcription factor GATA binding protein 3 (GATA-3) and connexin 32 (Cx32) in cultured gastric mucosa cells, and their association with each other. GES-1 cells were co-cultured with East Asian type cytotoxin-associated gene A+ H. pylori in the H. pylori group, and without H. pylori culture in the control group. Additionally, Mongolian gerbils were gavaged with H. pylori, and later the gastric antrum tissues were collected. The GATA-3 and Cx32 mRNA and protein expression levels were detected by a reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The scratch labeling fluorescent dye tracer (SLDT) technique was used to detect the gap junctional intercellular communication (GJIC) function. GATA-3 small interfering RNA (siRNA) was transfected into BGC823 cells and its effect on Cx32 expression levels was detected. The impact of GATA-3 on Cx32 promoter transcriptional activity was detected using a dual luciferase reporter assay. The results revealed that H. pylori infection increased GATA-3 expression and decreased Cx32 expression in GES-1 cells and in animal gastric tissues compared with their respective controls, whilst in BGC823 cells, GATA-3 siRNA increased Cx32 expression compared with the control. In the SLDT experiment of GES-1 cells with H. pylori infection, the fluorescent dye was primarily limited to a single cell row close to the scratch, and only a limited amount of dye passing to the second cell row, indicating that the GJIC function was substantially reduced or absent compared with the control group, where the fluorescence dye transferred to the neighboring cells of 3-4 rows, indicating a stronger GJIC function comparatively. GATA-3 inhibited the expression of the luciferase reporter gene, compared with the controls, suggesting that GATA-3 inhibited the expression of Cx32 by binding to Cx32 promoter sites. These results indicated that H. pylori-increased GATA-3 expression, which downregulated Cx32 expression, may serve an important function in gastric carcinogenesis, and GATA-3 siRNA may serve a function in the prevention and treatment of gastric cancer.

GATA-3 augmentation down-regulates Connexin43 in Helicobacter pylori associated gastric carcinogenesis.

Helicobacter pylori (H. pylori) is a risk factor of gastric carcinoma, and inflammation with H.pylori infection has widely been suggested to trigger gastric carcinogenesis through "inflammation-carcinoma chain" (non-atrophic gastritis (NAG) → chronic atrophic gastritis (CAG) → intestinal metaplasia (IM) → dysplasia (DYS) and gastric carcinoma (GC)). Connexin43 (Cx43) is a major constituent of gap junction in normal gastric mucosa (NGM) and it is continuously down-regulated from normal gastric mucosa to precancerous lesions or ultimate gastric carcinoma, which shows novel target against gastric carcinoma by preventing the Cx43 decline. Our previous studies demonstrated that H. pylori infection in gastric mucosa down-regulates Cx43 expression, but its mechanism remains unknown. The transcriptional factor, GATA binding protein 3 (GATA-3) is the key to regulate adaptive immune response, which possibly relates to inflammation toward malignant transformation. Here the substantial rising of GATA-3 was screened by transcriptional factor microarray along the developmental stages of H. pylori associated gastric carcinoma. Moreover, the increased GATA-3 and inhibited Cx43 were confirmed in clinical specimens, Mongolian gerbils and normal gastric epithelial cell line GES-1 with H. pylori infection. GATA-3 silencing generated the Cx43 restoration both in intermediate differentiation gastric cancer cells BGC-803 and in H. pylori infected GES-1 cells. Dual-luciferase reporter assay further revealed the GATA-3 as one of Cx43 down-regulators by directly binding to its promoters. Together, the incremental GATA-3 is found in H. pylori associated gastric carcinogenesis, which is responsible for Cx43 inhibition as well.