Chitosan-Rapamycin Carbon Dots Alleviate Glaucomatous Retinal Injury by Inducing Autophagy to Promote M2 Microglial Polarization.

Introduction: Glaucoma is a prevalent cause of irreversible vision impairment, characterized by progressive retinal ganglion cells (RGCs) loss, with no currently available effective treatment. Rapamycin (RAPA), an autophagy inducer, has been reported to treat glaucoma in rodent models by promoting RGC survival, but its limited water solubility, systemic toxicity, and pre-treatment requirements hinder its potential clinical applications. Methods: Chitosan (CS)-RAPA carbon dot (CRCD) was synthesized via hydrothermal carbonization of CS and RAPA and characterized by transmission electron microscopy, Fourier transform infrared spectra, and proton nuclear magnetic resonance. In vitro assays on human umbilical cord vein endothelial and rat retinal cell line examined its biocompatibility and anti-oxidative capabilities, while lipopolysaccharide-stimulated murine microglia (BV2) assays measured its effects on microglial polarization. In vivo, using a mouse retinal ischemia/reperfusion (I/R) model by acute intraocular pressure elevation, the effects of CRCD on visual function, RGC apoptosis, oxidative stress, and M2 microglial polarization were examined. Results: CRCD exhibited good water solubility and anti-oxidative capabilities, in the form of free radical scavenging. In vitro, CRCD was bio-compatible and lowered oxidative stress, which was also found in vivo in the retinal I/R model. Additionally, both in vitro with lipopolysaccharide-stimulated BV2 cells and in vivo with the I/R model, CRCD was able to promote M2 microglial polarization by activating autophagy, which, in turn, down-regulated pro-inflammatory cytokines, such as IL-1ß and TNF-α, as well as up-regulated anti-inflammatory cytokines, such as IL-4 and TGF-ß. All these anti-oxidative and anti-inflammatory effects ultimately aided in preserving RGCs, and subsequently, improved visual function. Discussion: CRCD could serve as a potential novel treatment strategy for glaucoma, via incorporating RAPA into CDs, in turn not only mitigating its toxic side effects but also enhancing its therapeutic efficacy.

Young Sca-1+ bone marrow stem cell-derived exosomes preserve visual function via the miR-150-5p/MEKK3/JNK/c-Jun pathway to reduce M1 microglial polarization.

BACKGROUND: Polarization of microglia, the resident retinal immune cells, plays important roles in mediating both injury and repair responses post-retinal ischemia-reperfusion (I/R) injury, which is one of the main pathological mechanisms behind ganglion cell apoptosis. Aging could perturb microglial balances, resulting in lowered post-I/R retinal repair. Young bone marrow (BM) stem cell antigen 1-positive (Sca-1+) cells have been demonstrated to have higher reparative capabilities post-I/R retinal injury when transplanted into old mice, where they were able to home and differentiate into retinal microglia. METHODS: Exosomes were enriched from young Sca-1+ or Sca-1- cells, and injected into the vitreous humor of old mice post-retinal I/R. Bioinformatics analyses, including miRNA sequencing, was used to analyze exosome contents, which was confirmed by RT-qPCR. Western blot was then performed to examine expression levels of inflammatory factors and underlying signaling pathway proteins, while immunofluorescence staining was used to examine the extent of pro-inflammatory M1 microglial polarization. Fluoro-Gold labelling was then utilized to identify viable ganglion cells, while H&E staining was used to examine retinal morphology post-I/R and exosome treatment. RESULTS: Sca-1+ exosome-injected mice yielded better visual functional preservation and lowered inflammatory factors, compared to Sca-1-, at days 1, 3, and 7 days post-I/R. miRNA sequencing found that Sca-1+ exosomes had higher miR-150-5p levels, compared to Sca-1- exosomes, which was confirmed by RT-qPCR. Mechanistic analysis found that miR-150-5p from Sca-1+ exosomes repressed the mitogen-activated protein kinase kinase kinase 3 (MEKK3)/JNK/c-Jun axis, leading to IL-6 and TNF-α downregulation, and subsequently reduced microglial polarization, all of which contributes to reduced ganglion cell apoptosis and preservation of proper retinal morphology. CONCLUSION: This study elucidates a potential new therapeutic approach for neuroprotection against I/R injury, via delivering miR-150-5p-enriched Sca-1+ exosomes, which targets the miR-150-5p/MEKK3/JNK/c-Jun axis, thereby serving as a cell-free remedy for treating retinal I/R injury and preserving visual functioning.

A novel fluorescent biosensor based on affinity-enhanced aptamer-peptide conjugate for sensitive detection of lead(II) in aquatic products.

Lead contamination is a major concern in food safety and, as such, many lead detection methods have been developed, especially aptamer-based biosensors. However, the sensitivity and environmental tolerance of these sensors require improvement. A combination of different types of recognition elements is an effective way to improve the detection sensitivity and environmental tolerance of biosensors. Here, we provide a novel recognition element, an aptamer-peptide conjugate (APC), to achieve enhanced affinity of Pb2+. The APC was synthesized from Pb2+ aptamers and peptides through clicking chemistry. The binding performance and environmental tolerance of APC with Pb2+ was studied through isothermal titration calorimetry (ITC); the binding constant (Ka) was 1.76*106 M-1, indicating that the APC's affinity was increased by 62.96% and 802.56% compared with the aptamers and peptides, respectively. Besides, APC demonstrated better anti-interference (K+) than aptamer and peptide. Through the molecular dynamics (MD) simulation, we found that more binding sites and stronger binding energy between APC with Pb2+are the reasons for higher affinity between APC with Pb2+. Finally, a carboxyfluorescein (FAM)-labeled APC fluorescent probe was synthesized and a fluorescent detection method for Pb2+ was established. The limit of detection of the FAM-APC probe was calculated to be 12.45 nM. This detection method was also applied to the swimming crab and showed great potential in real food matrix detection.

The class B heat shock factor HSFB1 regulates heat tolerance in grapevine.

Grape is a widely cultivated crop with high economic value. Most cultivars derived from mild or cooler climates may not withstand increasing heat stress. Therefore, dissecting the mechanisms of heat tolerance in grapes is of particular significance. Here, we performed comparative transcriptome analysis of Vitis davidii 'Tangwei' (heat tolerant) and Vitis vinifera 'Jingxiu' (heat sensitive) grapevines after exposure to 25°C, 40°C, or 45°C for 2 h. More differentially expressed genes (DEGs) were detected in 'Tangwei' than in 'Jingxiu' in response to heat stress, and the number of DEGs increased with increasing treatment temperatures. We identified a class B Heat Shock Factor, HSFB1, which was significantly upregulated in 'Tangwei', but not in 'Jingxiu', at high temperature. VdHSFB1 from 'Tangwei' and VvHSFB1 from 'Jingxiu' differ in only one amino acid, and both showed similar transcriptional repression activities. Overexpression and RNA interference of HSFB1 in grape indicated that HSFB1 positively regulates the heat tolerance. Moreover, the heat tolerance of HSFB1-overexpressing plants was positively correlated to HSFB1 expression level. The activity of the VdHSFB1 promoter is higher than that of VvHSFB1 under both normal and high temperatures. Promoter analysis showed that more TATA-box and AT~TATA-box cis-elements are present in the VdHSFB1 promoter than the VvHSFB1 promoter. The promoter sequence variations between VdHSFB1 and VvHSFB1 likely determine the HSFB1 expression levels that influence heat tolerance of the two grape germplasms with contrasting thermotolerance. Collectively, we validated the role of HSFB1 in heat tolerance, and the knowledge gained will advance our ability to breed heat-tolerant grape cultivars.

Natural variations of HSFA2 enhance thermotolerance in grapevine.

Heat stress limits growth and development of crops including grapevine which is a popular fruit in the world. Genetic variability in crops thermotolerance is not well understood. We identified and characterized heat stress transcription factor HSFA2 in heat sensitive Vitis vinifera 'Jingxiu' (named as VvHSFA2) and heat tolerant Vitis davidii 'Tangwei' (named as VdHSFA2). The transcriptional activation activities of VdHSFA2 are higher than VvHSFA2, the variation of single amino acid (Thr315Ile) in AHA1 motif leads to the difference of transcription activities between VdHSFA2 and VvHSFA2. Based on 41 Vitis germplasms, we found that HSFA2 is differentiated at coding region among heat sensitive V. vinifera, and heat tolerant Vitis davidii and Vitis quinquangularis. Genetic evidence demonstrates VdHSFA2 and VvHSFA2 are positive regulators in grape thermotolerance, and the former can confer higher thermotolerance than the latter. Moreover, VdHSFA2 can regulate more target genes than VvHSFA2. As a target gene of both VdHSFA2 and VvHSFA2, overexpression of MBF1c enhanced the grape thermotolerance whereas dysfunction of MBF1c resulted in thermosensitive phenotype. Together, our results revealed that VdHSFA2 confers higher thermotolerance than VvHSFA2, and MBF1c acts as their target gene to induce thermotolerance. The VdHSFA2 may be adopted for molecular breeding in grape thermotolerance.

Improvement of Photosynthesis by Biochar and Vermicompost to Enhance Tomato (Solanum lycopersicum L.) Yield under Greenhouse Conditions.

Chlorophyll fluorescence is an important tool in the study of photosynthesis and its effect on the physiological indicators of crop growth is worth exploring. The trial was conducted to investigate the effect of biochar (CK, 0%; BA3, 3%; BA5, 5%; by mass of soil) and vermicompost (VA3, 3%; VA5, 5%) on photosynthesis, chlorophyll fluorescence, and tomato yield under greenhouse condition. Results revealed that photosynthetic parameters and chlorophyll fluorescence traits of BA3, VA3, BA5, and VA5 were significantly higher than those of CK, and the improvement of vermicompost was more effective than biochar at the same application rate. VA3 treatment had the highest net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), variable fluorescence (Fv), maximum fluorescence (Fm), PSII maximum photochemical efficiency (Fv/Fm), PSII potential photochemical activity (Fv/Fo), absorption flux per cross section (CS; ABC/CSm), trapped energy flux per CS (TRo/CSm), and electron transport flux per CS (ETo/CSm), which increased by 49%, 65%, 17%, 12%, 4%, 25%, 10%, 15%, and 30%, respectively, compared with CK. The study also found that BA and VA rates could effectively improve tomato yield and water use efficiency (WUE). The yield under BA3, VA3, BA5, and VA5 treatments was 21%, 33%, 23%, and 25% higher than that under CK, and the WUE increased from 31.2 kg·m-3 under CK to 41.4 kg·m-3 under VA3. Pearson correlation analysis indicated that the increment of photosynthesis showed a highly significant correlation with Fv/Fo, ABC/CSm, TRo/CSm, and ETo/CSm and enhanced the light energy absorbed, trapped, and transported per CS of plant leaves, thereby contributing to the increase in tomato yield. Therefore, for one-season tomato production, the application of 3% vermicompost was considered economical with regard to improving photosynthesis, enhancing WUE, and increasing tomato yield.

Opposite impact of DOM on ROS generation and photoaging of aromatic and aliphatic nano- and micro-plastic particles.

Dissolved organic matter (DOM) plays a significant role in the photochemical behavior of nano- and micro-plastic particles (NPs/MPs). We investigated the influence of DOM on the mechanism on the photoaging of NPs/MPs with different molecular structures under UV365 irradiation in water. DOM components used in this study are mainly humic acid and fulvic acid. The results showed that DOM promoted the weathering of aliphatic NPs/MPs (polypropylene (PP)), but inhibited or had only a minor effect on the photoaging of aromatic NPs/MPs (polystyrene (PS) NPs/MPs, carboxyl-modified PS NPs, amino-modified PS NPs, and polycarbonate MPs). NPs with a large surface area may adsorb sufficient DOM on the particle surfaces through π-π interactions, which competes with NPs for photon absorption sites, thus, can delay the photoaging of PS NPs. Aromatic MPs may release phenolic compounds that quench •OH, thereby weakening the photoaging process. For aliphatic MPs, the detection of peracid, aldehyde, and ketone groups on the polymer surface indicated that DOM promoted weathering of PP MPs, which was primarily because the generation of •OH due to DOM photolysis may attack the polymer by C-C bond cleavage and hydrogen extraction reactions. This study provides insight into the UV irradiation weathering process of NPs/MPs of various compositions and structures, which are globally distributed in water.

Strain-Controlled Fatigue Behavior and Microevolution of 316L Stainless Steel under Cyclic Shear Path.

Based on the twin bridge shear specimen, the cyclic shear experiments were performed on 1.2 mm thin plates of 316L metastable austenitic stainless steel with different strain amplitudes from 1 to 5% at ambient temperature. The fatigue behavior of 316L stainless steel under the cyclic shear path was studied, and the microscopic evolution of the material was analyzed. The results show that the cyclic stress response of 316L stainless steel exhibited cyclic hardening, saturation and cyclic softening, and the fatigue life is negatively correlated with the strain amplitude. The microstructure was analyzed by using electron back-scattered diffraction (EBSD). It was found that grain refinement and martensitic transformation during the deformation process led to rapid crack expansion and reduced the fatigue life of 316L.

Age-related visual impairments and retinal ganglion cells axonal degeneration in a mouse model harboring OPTN (E50K) mutation.

Retinal ganglion cells (RGCs) axons are the signal carriers of visual information between retina and brain. Therefore, they play one of the important roles affected in many optic neurodegenerative diseases like glaucoma. Among the genetic risks associated with glaucoma, the E50K mutation in the Optineurin (OPTN) gene are known to result in glaucoma in the absence of increased intraocular pressure (IOP), whereas the relevant pathological mechanism and neurological issues remain to be further investigated. In this study, the OPTN (E50K) mutant mouse model was established through CRISPR/Cas9-mediated genome editing, and aging-related RGCs loss and the visual dysfunction were identified. In E50K mice 16 months old, the axonal transport decreased comparing to wild-type (WT) mice at the same age. Furthermore, results of electron microscopy demonstrated significant morphological anomaly of mitochondria in RGCs axons of young E50K mice 3 months old, and these changes were aggravated with age. These indicated that the damaged mitochondria-associated dysfunction of RGCs axon should play an etiological role in glaucoma as an age-related outcome of OPTN (E50K) mutation. The findings of this study have potential implications for the targeted prevention and treatment of NTG.

A Novel Aptamer-Imprinted Polymer-Based Electrochemical Biosensor for the Detection of Lead in Aquatic Products.

Lead contamination in aquatic products is one of the main hazard factors. The aptasensor is a promising detection method for lead ion (Pb(II)) because of its selectivity, but it is easily affected by pH. The combination of ion-imprinted polymers(IIP) with aptamers may improve their stability in different pH conditions. This paper developed a novel electrochemical biosensor for Pb(II) detection by using aptamer-imprinted polymer as a recognition element. The glassy carbon electrode was modified with gold nanoparticles and aptamers. After the aptamer was induced by Pb(II) to form a G-quadruplex conformation, a chitosan-graphene oxide was electrodeposited and cross-linked with glutaraldehyde to form an imprint layer, improving the stability of the biosensor. Under the optimal experimental conditions, the current signal change (∆I) showed a linear correlation of the content of Pb(II) in the range of 0.1-2.0 µg/mL with a detection limit of 0.0796 µg/mL (S/N = 3). The biosensor also exhibited high selectivity for the determination of Pb(II) in the presence of other interfering metal ion. At the same time, the stability of the imprinted layer made the sensor applicable to the detection environment with a pH of 6.4-8.0. Moreover, the sensor was successfully applied to the detection of Pb(II) in mantis shrimp.

Neuroprotective effects of bone marrow Sca-1+ cells against age-related retinal degeneration in OPTN E50K mice.

Glaucoma is characterized by retinal ganglion cell (RGC) death, the underlying mechanisms of which are still largely unknown. An E50K mutation in the Optineurin (OPTN) gene is a leading cause of normal-tension glaucoma (NTG), which directly affects RGCs in the absence of high intraocular pressure and causes severe glaucomatous symptoms in patients. Bone marrow (BM) stem cells have been demonstrated to play a key role in regenerating damaged tissue during ageing and disease through their trophic effects and homing capability. Here, we separated BM stem cells into Sca-1+ and Sca-1- cells and transplanted them into lethally irradiated aged OPTN E50K mice to generate Sca-1+ and Sca-1- chimaeras, respectively. After 3 months of BM repopulation, we investigated whether Sca-1+ cells maximized the regenerative effects in the retinas of NTG model mice with the OPTN E50K mutation. We found that the OPTN E50K mutation aggravated age-related deficiency of neurotrophic factors in both retinas and BM during NTG development, leading to retinal degeneration and BM dysfunction. Sca-1+ cells from young healthy mice had greater paracrine trophic effects than Sca-1- cells and Sca-1+ cells from young OPTN E50K mice. In addition, Sca-1+ chimaeras demonstrated better visual functions than Sca-1- chimaeras and untreated OPTN E50K mice. More Sca-1+ cells than Sca-1- cells were recruited to repair damaged retinas and reverse visual impairment in NTG resulting from high expression levels of neurotrophic factors. These findings indicated that the Sca-1+ cells from young, healthy mice may have exhibited an enhanced ability to repair retinal degeneration in NTG because of their excellent neurotrophic capability.

Proteomic analysis of aged and OPTN E50K retina in the development of normal tension glaucoma.

Progressive degeneration of retinal ganglion cells (RGCs) is a major characteristic of glaucoma, whose underlying mechanisms are still largely unknown. An E50K mutation in the Optineurin (OPTN) gene is a leading cause of normal tension glaucoma (NTG), directly affecting RGCs without high intraocular pressure and causing severe glaucomatous symptoms in clinical settings. A systematic analysis of the NTG mouse model is crucial for better understanding of the underlying pathological mechanisms for glaucoma. To elucidate proteomic and biochemical pathway alterations during NTG development, we established an OPTN E50K mutant mouse model through CRISPR/Cas9. Retinal proteins from resulting mice exhibiting glaucomatous phenotypes were subject to tandem mass tag-labeled quantitative proteomics and then analyzed through bioinformatics methods to characterize the molecular and functional signatures of NTG. We identified 6364 quantitative proteins in our proteomic analysis. Bioinformatics analysis revealed that OPTN E50K mice experienced protein synthesis dysregulation, age-dependent energy defects and autophagy-lysosome pathway dysfunction. Certain biological features, including amyloid deposition, RNA splicing, microglia activation and reduction of crystallin production, were similar to Alzheimer's disease. Our study is the first to describe proteomic and biochemical pathway alterations in NTG pathogenesis during disease advancement. Several proteomic signatures overlapped with retinal changes found in the ad mice model, suggesting the presence of common mechanisms between age-related degenerative disorders, as well as prospective new targets for diagnostic and therapeutic strategies.

The E50K optineurin mutation impacts autophagy-mediated degradation of TDP-43 and leads to RGC apoptosis in vivo and in vitro.

The glaucoma-associated E50K mutation in optineurin (OPTN) is known to affect autophagy and cause the apoptosis of retinal ganglion cells (RGCs), but the pathogenic mechanism remains unclear. In this study, we investigated whether the OPTN (E50K) mutation caused TDP-43 aggregation by disrupting autophagy in vivo and in vitro. OPTN (E50K) mutant mice were generated and analysed for genotype and phenotype. Adeno-associated virus type 2 vectors containing either GFP only, GFP-tagged wild-type OPTN or GFP-tagged E50K-mutated OPTN were used to transfect R28 cells. Loss of RGCs decreased retinal thickness and visual impairment were observed in OPTN (E50K) mice compared with WT mice. Moreover, overexpression of E50K OPTN induced R28 cell apoptosis. Increased p62/SQSTM1 and LC3-II levels indicated that autophagic flux was inhibited and contributed to TDP-43 aggregation in vivo and in vitro. We found that rapamycin effectively reduced the aggregation of TDP-43 in OPTN (E50K) mice and decreased the protein levels of p62/SQSTM1 and the autophagic marker LC3-II. Moreover, rapamycin increased the RGC number and visual function of E50K mice. In addition, we also observed increased cytoplasmic TDP-43 in the spinal cord and motor dysfunction in 24-month-old OPTN (E50K) mice, indicating that TDP-43 accumulation may be the common pathological mechanism of glaucoma and amyotrophic lateral sclerosis (ALS). In conclusion, the disruption of autophagy by OPTN (E50K) affected the degradation of TDP-43 and may play an important role in OPTN (E50K)-mediated glaucomatous retinal neurodegeneration.

New insights into the heat responses of grape leaves via combined phosphoproteomic and acetylproteomic analyses.

Heat stress is a serious and widespread threat to the quality and yield of many crop species, including grape (Vitis vinifera L.), which is cultivated worldwide. Here, we conducted phosphoproteomic and acetylproteomic analyses of leaves of grape plants cultivated under four distinct temperature regimes. The phosphorylation or acetylation of a total of 1011 phosphoproteins with 1828 phosphosites and 96 acetyl proteins with 148 acetyl sites changed when plants were grown at 35 °C, 40 °C, and 45 °C in comparison with the proteome profiles of plants grown at 25 °C. The greatest number of changes was observed at the relatively high temperatures. Functional classification and enrichment analysis indicated that phosphorylation, rather than acetylation, of serine/arginine-rich splicing factors was involved in the response to high temperatures. This finding is congruent with previous observations by which alternative splicing events occurred more frequently in grapevine under high temperature. Changes in acetylation patterns were more common than changes in phosphorylation patterns in photosynthesis-related proteins at high temperatures, while heat-shock proteins were associated more with modifications involving phosphorylation than with those involving acetylation. Nineteen proteins were identified with changes associated with both phosphorylation and acetylation, which is consistent with crosstalk between these posttranslational modification types.

Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature.

Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels.