Exosomal lncRNA-MIAT promotes neovascularization via the miR-133a-3p/MMP-X1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.

Real-time detection of human telomerase DNA synthesis by multiplexed single-molecule FRET.

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.

Dynamics of release factor recycling during translation termination in bacteria.

In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

The Traditional Chinese Medicine Houttuynia cordata Thunb decoction alters intestinal barrier function via an EGFR dependent MAPK (ERK1/2) signalling pathway.

BACKGROUND: A traditionally prepared aqueous extract (= decoction) of Houttuynia cordata Thunb (Yu xing cao) (HC) is widely used in Traditional Chinese Medicine (TCM) to treat inflammatory disease. Previous chemical and biological studies on HC have mainly focused on organic extracts rather than the aqueous decoction, which is the traditional formulation. PURPOSE: The study aimed to investigate whether the chemical composition of HC aqueous decoction (HCD) varies with geographical sourcing, to investigate the mechanism of action of HCD, and to determine if chemical variation impacts on HCDs anti-inflammatory activity. METHOD: Sixteen samples of HC were purchased from Sichuan, Hubei and Anhui provinces in the People's Republic of China (PRC) and were prepared by the traditional decoction method to yield their corresponding HCDs. A Quality Control (QC) sample was prepared by combining individual HCD extracts. HCDs were analysed by Nuclear Magnetic Resonance (NMR) and High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS). The anti-inflammatory activities associated with intestinal barrier function of HCD were studied by tumor necrosis factor-α (TNF-α) activated Caco-2 monolayers in vitro and in vivo using Dextran Sulfate Sodium (DSS)-induced murine colitis. Proteins involved in inflammation, mRNA levels, disease severity scores, and histology involved in intestinal inflammation were analysed. RESULTS: HCD samples exhibited different chemical fingerprints and three regional outliers were identified by Principal Component Analysis (PCA). Fifteen phytochemical metabolites were identified and quantified. HCD showed in vitro anti-inflammatory activity, enhancing zonula occludens-1 (ZO-1), occludin, interleukin (IL)-10 and decreasing IL-1ß, IL-6 and epidermal growth factor receptor (EGFR) via an EGFR-dependent mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK 1/2) signaling pathway. This beneficial effect on intestinal inflammation was also seen in the in vivo colitis model at a molecular level in colonic tissues. CONCLUSION: This study shows that the test HCDs were chemically different, resulting in different levels of activity on intestinal barrier function and inflammation. Moreover, a "Daodi" product showed the greatest biological activity in this study, thus validating the importance of the "Daodi" quality material in TCM and supporting the traditional used of HCD for the treatment of inflammation.

Network Pharmacology and Molecular Docking Elucidate the Underlying Pharmacological Mechanisms of the Herb Houttuynia cordata in Treating Pneumonia Caused by SARS-CoV-2.

Used in Asian countries, including China, Japan, and Thailand, Houttuynia cordata Thumb (H. cordata; Saururaceae, HC) is a traditional herbal medicine that possesses favorable antiviral properties. As a potent folk therapy used to treat pulmonary infections, further research is required to fully elucidate the mechanisms of its pharmacological activities and explore its therapeutic potential for treating pneumonia caused by SARS-CoV-2. This study explores the pharmacological mechanism of HC on pneumonia using a network pharmacological approach combined with reprocessing expression profiling by high-throughput sequencing to demonstrate the therapeutic mechanisms of HC for treating pneumonia at a systemic level. The integration of these analyses suggested that target factors are involved in four signaling pathways, including PI3K-Akt, Jak-STAT, MAPK, and NF-kB. Molecular docking and molecular dynamics simulation were applied to verify these results, indicating a stable combination between four metabolites (Afzelin, Apigenin, Kaempferol, Quercetin) and six targets (DPP4, ELANE, HSP90AA1, IL6, MAPK1, SERPINE1). These natural metabolites have also been reported to bind with ACE2 and 3CLpro of SARS-CoV-2, respectively. The data suggest that HC exerts collective therapeutic effects against pneumonia caused by SARS-CoV-2 and provides a theoretical basis for further study of the active drug-like ingredients and mechanism of HC in treating pneumonia.

Analysis of Microcirculation Changes in the Macular Area and Para-Optic Disk Region After Implantable Collamer Lens Implantation in Patients With High Myopia.

Myopia has become an important public health problem to be solved urgently. Posterior chamber phakic implantable Collamer lens (ICL) implantation is one of the latest and safest products for myopia correction worldwide. This prospective cross-sectional case series aimed to observe changes in the macular retinal thickness, retinal nerve fiber layer (RNFL) thickness of para-optic disk region, and blood flow density after posterior ICL implantation in patients with high myopia using optical coherence tomography angiography (OCTA). A total of 67 eyes of 67 patients with high myopia, who underwent ICL implantation at The Affiliated Eye Hospital of Nanjing Medical University from January 2020 and December 2020, were included. The spherical equivalent (SE) of the operative eyes was >-6.00 D. The changes in vision, intraocular pressure (IOP), SE, and vault were observed pre-operatively, and follow-up were performed 1 week, 1 month, and 3 months. OCTA was used to observe the changes in the CRT, retinal thickness of paracentral fovea, FAZ, superficial and deep retinal blood flow density in the macular area, RNFL thickness of para-optic disk region, and blood flow density before and after ICL implantation. The uncorrected distance visual acuity (UDVA) and best corrected distance visual acuity (CDVA) of the patients post-operation were significantly improved (P < 0.001). The IOP increased in comparison with other time points at 1 week post-operation (P < 0.05). There were no significant changes in CRT post-operation. The retinal thickness in the upper, lower, nasal, and temporal quadrants of the paracentral fovea increased significantly at 1 month and 3 months post-operation (P < 0.05). The FAZ area at all postoperative time points were decreased (P < 0.001). At 3 months post-operation, the blood flow density of the superficial and deep retinal layers in the upper, lower, and nasal macular area were significantly reduced (P < 0.05). At 1 month post-operation, the RNFL thickness in the temporal para-optic disk region and blood flow density were significantly reduced (P = 0.001 and P < 0.05, respectively). ICL implantation for highly myopic eyes led to an increase of the retinal thickness in the upper, lower, nasal, and temporal regions of the paracentral fovea; reduction of RNFL thickness in the temporal area of para-optic disk; decrease in FAZ area; and decrease in the blood flow density of some deep and superficial retinal layers as well as that of the temporal para-optic disk region.

eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA.

Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.

Hesperetin's health potential: moving from preclinical to clinical evidence and bioavailability issues, to upcoming strategies to overcome current limitations.

Flavonoids are common in the plant kingdom and many of them have shown a wide spectrum of bioactive properties. Hesperetin (Hst), the aglycone form of hesperidin, is a great example, and is the most abundant flavonoid found in Citrus plants. This review aims to provide an overview on the in vitro, in vivo and clinical studies reporting the Hst pharmacological effects and to discuss the bioavailability-related issues. Preclinical studies have shown promising effects on cancer, cardiovascular diseases, carbohydrate dysregulation, bone health, and other pathologies. Clinical studies have supported the Hst promissory effects as cardioprotective and neuroprotective agent. However, further well-designed clinical trials are needed to address the other Hst effects observed in preclinical trials, as well as to a more in-depth understanding of its safety profile.

Resveratrol-Based Nanoformulations as an Emerging Therapeutic Strategy for Cancer.

Resveratrol is a polyphenolic stilbene derivative widely present in grapes and red wine. Broadly known for its antioxidant effects, numerous studies have also indicated that it exerts anti-inflammatory and antiaging abilities and a great potential in cancer therapy. Regrettably, the oral administration of resveratrol has pharmacokinetic and physicochemical limitations such as hampering its effects so that effective administration methods are demanding to ensure its efficiency. Thus, the present review explores the published data on the application of resveratrol nanoformulations in cancer therapy, with the use of different types of nanodelivery systems. Mechanisms of action with a potential use in cancer therapy, negative effects, and the influence of resveratrol nanoformulations in different types of cancer are also highlighted. Finally, the toxicological features of nanoresveratrol are also discussed.

Mechanisms that ensure speed and fidelity in eukaryotic translation termination.

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bound together to recognize stop codons rapidly and elicit termination through a tightly regulated, multistep process that resembles transfer RNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

Structural basis for the transition from translation initiation to elongation by an 80S-eIF5B complex.

Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNAiMet on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNAiMet immediately before transition into elongation. The structure uncovers an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.

Demographic and motivational factors affecting the whole-body donation programme in Nanjing, China: a cross-sectional survey.

OBJECTIVES: To investigate the demographics and motivations of whole-body donors in China, and help suggest a solution to the problem of low body donation numbers. DESIGN: A cross-sectional study on body donors in China. Demographic analysis of the donating information of deceased donors and in-depth interviews of potential body donors. SETTING: Eleven districts in Nanjing, China. PARTICIPANTS: Deceased whole-body donors who had donated their bodies to the body donation receiving station of Nanjing Medical University between 1 July 2009 and 30 June 2019 (n=835), and living registered whole-body donors (n=68). RESULTS: Among the whole-body donor population, the numbers of males, people older than 65 years and those working as teachers, government officials, medical staff and farmers were significantly higher than those of the general Nanjing population. Donors with an education level of college or above accounted for nearly half of the deceased donors, and considered donating their bodies earlier in their lives than others. Cancer and heart disease were the major causes of death among donors. Interviews of the 68 living donors revealed the following major motivations for the decision to donate: to support medical education; to reduce their children's funeral burden; no longer holding traditional Chinese views on life and death; influence by role models and annoyance at complex funeral ceremonies. CONCLUSIONS: Older people, people with an education level of college or above, labourers, teachers, government officials and farmers are the major groups that donate their bodies. Although people's motivations for donation are complex, their desire to support medical education is the most prevalent motivation. By helping focus on target groups for promotional messaging and identifying their prime motivations, this study's findings can provide a reference for promoting body donation in China.

Discrimination of Mentha species grown in different geographical areas of Algeria using 1H-NMR-based metabolomics.

1H-NMR-based metabolomics have been applied to identify potential NMR-markers and biomarkers capable of distinguishing, qualifying and classifying three Mentha species:- Mentha pulegium L., Mentha × rotundifolia (L.) Huds., Mentha spicata L., and their ecotypes. Samples of the 3 species were collected in seven different locations in Algeria, with the aim to establish a quality control protocol based on the use of NMR fingerprint profiles of polar extracts. NMR data indicate that the identification of the Mentha genus can be confirmed by the presence of the doublet proton signals with identical coupling constants at δ 7.49 (d, 15.9 Hz) and δ 6.29 (d, 15.9 Hz); these correspond to the protons of the double-bond conjugated to the ester group of rosmarinic acid, a bioactive compound found in all three species. Differences in NMR proton chemical shifts and/or signal intensities were clearly demonstrated on the orthogonal projections to latent structures discriminating analysis (OPLS-DA). Several potential biomarkers discriminating the three Mentha species were originated using S-plots, loading score plots, NMR data analysis and literature search. These discriminating signals point to glycosylated flavonols, oxygenated terpenoids and hydrocarbon terpenoids to distinguish M. pulegium, M. × rotundifolia and M. spicata, respectively. Within the same species, Principal Component Analysis (PCA) scores clearly discriminated the metabolite content according to regions in which the plants were grown. The 6 zones in which Mentha pulegium samples were harvested were clearly separated along either or both PC1 and PC2; by contrast, the harvesting locations were divided into two groups along PC1 for both M. × rotundifolia and M. spicata. The total antioxidant activity of the Mentha species was impacted by the abiotic factors of the different regions.

Dynamics of the context-specific translation arrest by chloramphenicol and linezolid.

Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide-bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not substantially alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site transfer RNA to undergo repeated rounds of dissociation and nonproductive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.

eIF5B gates the transition from translation initiation to elongation.

Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.

RACK1 on and off the ribosome.

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein (RP) implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as a target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any RP from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular nonessentiality from yeast to humans, RACK1 readily reassociates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight into the biochemical basis of RPs exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

Kinetics of d-Amino Acid Incorporation in Translation.

Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow d-Phe incorporation was unexpectedly very efficient in competition with incorporation of the l isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an l-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.