RALY participates in nerve trauma-induced nociceptive hypersensitivity through triggering Eif4g2 gene expression in primary sensory neurons.

BACKGROUND AND PURPOSE: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. METHODS AND RESULTS: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (Eif4g2) mRNA and Eif4g2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA up-regulated the expression of Eif4g2 mRNA and Eif4g2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of Eif4g2 gene and triggered its transcriptional activity. CONCLUSION AND IMPLICATIONS: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering Eif4g2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

Specific inhibition of TET1 in the spinal dorsal horn alleviates inflammatory pain in mice by regulating synaptic plasticity.

DNA demethylation mediated by ten-eleven translocation 1 (TET1) is a critical epigenetic mechanism in which gene expression is regulated via catalysis of 5-methylcytosine to 5-hydroxymethylcytosine. Previously, we demonstrated that TET1 is associated with the genesis of chronic inflammatory pain. However, how TET1 participates in enhanced nociceptive responses in chronic pain remains poorly understood. Here, we report that conditional knockout of Tet1 in dorsal horn neurons via intrathecal injection of rAAV-hSyn-Cre in Tet1fl/fl mice not only reversed the inflammation-induced upregulation of synapse-associated proteins (post-synaptic density protein 95 (PSD95) and synaptophysin (SYP)) in the dorsal horn but also ameliorated abnormalities in dendritic spine morphology and alleviated pain hypersensitivities. Pharmacological blockade of TET1 by intrathecal injection of a TET1-specific inhibitor-Bobcat 339-produced similar results, as did knockdown of Tet1 by intrathecal injection of siRNA. Thus, our data strongly suggest that increased TET1 expression during inflammatory pain upregulates the expression of multiple synapse-associated proteins and dysregulates synaptic morphology in dorsal horn neurons, suggesting that Tet1 may be a potential target for analgesic strategies.

Nickel/photoredox dual catalyzed arylalkylation of nonactivated alkenes.

Alkene dicarbofunctionalization is an efficient strategy and operation-economic fashion for introducing complexity in molecules. A nickel/photoredox dual catalyzed arylalkylation of nonactivated alkenes for the simultaneous construction of one C(sp3)-C(sp3) bond and one C(sp3)-C(sp2) bond has been developed. The mild catalytic method provided valuable indanethylamine derivatives with wide substrate scope and good functional group compatibility. An enantioselective dicarbofunctionalization was also achieved with pyridine-oxazoline as a ligand. The efficiency of metallaphotoredox dicarbofunctionalization was demonstrated for the concise synthesis of pharmaceutically active compounds.

Clinical Analysis of Repeat Penetrating Keratoplasty in Children.

(1) Background: To analyze the indications, graft survival, and graft failure-related risk factors of repeat penetrating keratoplasty (RPK) in children. (2) Methods: In this case series, children younger than 12 years who received RPK at Beijing Tongren Hospital were reviewed. The indications for RPK, postoperative complications, and graft survival were analyzed. The analysis of the potential variables associated with graft survival was performed using Cox proportional hazards regression. (3) Results: A total of 30 RPK eyes of 29 children were included in this study. The mean follow-up time was 26.98 ± 18.75 months. The most common indication for RPK was a vascularized corneal scar (86.67%). Postoperative complications occurred in 27 eyes (90%), including immune rejection (46.67%), epithelial defects (36.67%), and glaucoma (26.67%). About 60% of the regrafts remained clear one year after RPK, while the overall graft survival rate was 30% at the last visit. The most common cause of regraft failure was irreversible immune rejection (8/21). The significant risks of graft failure included an age of less than 60 months at surgery (p = 0.009), corneal vascularization (p = 0.018), and a postoperative epithelial defect (p = 0.037). (4) Conclusions: A vascularized corneal scar is the most common indication of RPK in children. Immune rejection is the most prevalent complication, and irreversible immune rejection always causes regraft failure.

Downregulation of ciRNA-Kat6b in dorsal spinal horn is required for neuropathic pain by regulating Kcnk1 in miRNA-26a-dependent manner.

AIMS: Nerve injury-induced maladaptive changes in gene expression in the spinal neurons are essential for neuropathic pain genesis. Circular RNAs (ciRNA) are emerging as key regulators of gene expression. Here, we identified a nervous-system-tissues-specific ciRNA-Kat6 with conservation in humans and mice. We aimed to investigate whether and how spinal dorsal horn ciRNA-Kat6b participates in neuropathic pain. METHODS: Unilateral sciatic nerve chronic constrictive injury (CCI) surgery was used to prepare the neuropathic pain model. The differentially expressed ciRNAs were obtained by RNA-Sequencing. The identification of nervous-system-tissues specificity of ciRNA-Kat6b and the measurement of ciRNA-Kat6b and microRNA-26a (miRNA-26a) expression level were carried out by quantitative RT-PCR. The ciRNA-Kat6b that targets miRNA-26a and miRNA-26a that targets Kcnk1 were predicted by bioinformatics analysis and verified by in vitro luciferase reports test and in vivo experiments including Western-blot, immunofluorescence, and RNA-RNA immunoprecipitation. The correlation between neuropathic pain and ciRNA-Kat6b, miRNA-26a, or Kcnk1 was examined by the hypersensitivity response to heat and mechanical stimulus. RESULTS: Peripheral nerve injury downregulated ciRNA-Kat6b in the dorsal spinal horn of male mice. Rescuing this downregulation blocked nerve injury-induced increase of miRNA-26a, reversed the miRNA-26a-triggered decrease of potassium channel Kcnk1, a key neuropathic pain player, in the dorsal horn, and alleviates CCI-induced pain hypersensitivities. On the contrary, mimicking this downregulation increased the miRNA-26a level and decreased Kcnk1 in the spinal cord, resulting in neuropathic pain-like syndrome in naïve mice. Mechanistically, the downregulation of ciRNA-Kat6b reduced the accounts of miRNA-26a binding to ciRNA-Kat6b, and elevated the binding accounts of miRNA-26a to the 3' untranslated region of Kcnk1 mRNA and degeneration of Kcnk1 mRNA, triggering in the reduction of KCNK1 protein in the dorsal horn of neuropathic pain mice. CONCLUSION: The ciRNA-Kat6b/miRNA-26a/Kcnk1 pathway in dorsal horn neurons regulates the development and maintenance of neuropathic pain, ciRNA-Kat6b may be a potential new target for analgesic and treatment strategies.

Epidemiologic characteristics and the change of surgical methods of pterygium and pseudopterygium from 2013 to 2019 in China: A retrospective analysis.

Introduction: Pterygium is a common multifactorial external eye disease, which causes various ocular symptoms and negatively affects appearance. The aim of this study was to analyze the epidemiological characteristics and the change of surgical methods of pterygium and pseudopterygium in China from 2013 to 2019. Materials and methods: This study was a hospital-based nationwide retrospective study to estimate the epidemiologic characteristics and the change of surgical methods of pterygium and pseudopteygium in China from 2013 to 2019. The data was extracted from the Hospital Quality Monitoring System (HQMS) database. The diagnosis was based on the tenth revision of the International Classification of Diseases (ICD-10) code. Results: Our study included 1,007,800 pterygium and 2,681 pseudopteygium inpatients. From 2013 to 2019, the proportion of pterygium and pseudopterygium patients who underwent surgery, among all ophthalmology inpatients, increased from 3.3% in 2013 to 7.84% in 2019. The male-female ratio of surgically treated pterygium and pseudopterygium is 1:1.8 and 1.6:1 respectively. Among all age groups, the hospitalized pterygium patients who received surgery were mainly 60-69 years old, accounting for 36.53%. The pseudopterygium patients who received surgery were mostly 50-59 years old, accounting for 24.02%. Among the 31 provinces of mainland China, Yunnan Province has the highest proportion of pterygium patients treated surgically (6.40%), while Shanghai has the highest proportion of pseudopterygium patients treated surgically (12.98%). The most common occupation of participants in the study was farmer, accounting for 47.62% and 28.53%, respectively. During the study period, the application of autologous stem cell transplantation increased year by year, and became the first choice for pterygium and pseudopterygium surgery. Discussion: This study was the first to describe the epidemiological characteristics and surgical methods of hospitalized pterygium and pseudopterygium patients in China. This study provides important information for better diagnosis, treatment and prevention of pterygium and pseudopterygium.

Astragalus Polysaccharide Promotes Doxorubicin-Induced Apoptosis by Reducing O-GlcNAcylation in Hepatocellular Carcinoma.

The toxicity and side effects of chemotherapeutic drugs remain a crucial obstacle to the clinical treatment of hepatocellular carcinoma (HCC). Identifying combination therapy from Chinese herbs to enhance the sensitivity of tumors to chemotherapeutic drugs is of particular interest. Astragalus polysaccharide (APS), one of the natural active components in Astragalus membranaceus, has been reported to exhibit anti-tumor properties in diverse cancer cell lines. The aim of this study was to determine the effect of APS on Doxorubicin (Dox)-induced apoptosis in HCC and the underlying mechanism. The results showed that APS dose-dependently promoted Dox-induced apoptosis and enhanced endoplasmic reticulum (ER) stress. Additionally, APS decreased the mRNA level and protein stability of O-GlcNAc transferase (OGT), and increased the O-GlcNAcase (OGA) expression. Furthermore, OGT lentiviral transfection or PugNAc (OGA inhibitor) treatment reversed the ER stress and apoptosis induced by the combination of Dox and APS. A xenograft tumor mouse model confirmed that the combination of APS and Dox showed an advantage in inhibiting tumor growth in vivo. These findings suggested that APS promoted Dox-induced apoptosis in HCC cells through reducing the O-GlcNAcylation, which led to the exacerbation of ER stress and activation of apoptotic pathways.

SLAMF7/STAT6 Pathway Inhibits Innate Immune Response in Late-Stage Human Acanthamoeba Keratitis: A Comparative Transcriptome Analysis.

Acanthamoeba keratitis (AK) is a blinding corneal infection caused by the protozoan Acanthamoeba. The long-term course of AK suggests the host immunity could not kill Acanthamoeba rapidly. The immune status is still unclear in the late stage of AK. The comparative transcriptome analysis was made based on the bulk RNA sequencing of cornea tissues from AK patients and donors. Differentially expressed genes and enriched signaling pathways were calculated. CIBERSORT algorithm was used for immune infiltration analysis of cornea tissue between AK and normal controls. A total of 2668 differentially expressed genes, including 1477 upregulated genes and 1191 downregulated genes, were detected. Gene Ontology analysis revealed that the pathways were significantly enriched in leukocyte migration, regulation of T-cell activation, the external side of plasma membrane, collagen-containing extracellular matrix, immune receptor activity, and cytokine binding. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the pathways were significantly enriched in the cytokine-cytokine receptor interaction, hematopoietic cell lineage, and Staphylococcus aureus infection pathway. The immune infiltration profiles varied little between AK and normal controls. Compared with normal tissue, cornea tissue of AK contained a higher proportion of M0 macrophages and CD8 T cells, while resting memory CD4 T cells contributed to a relatively lower portion (p < 0.05). Finally, the expression levels of cell markers and SLAMF7/STAT6 pathway were confirmed by histopathology examinations, RT-qPCR, and Western blot.

Microglial P2Y12 Signaling Contributes to Cisplatin-induced Pain Hypersensitivity via IL-18-mediated Central Sensitization in the Spinal Cord.

Administration of cisplatin and other chemotherapy drugs is crucial for treating tumors. However, cisplatin-induced pain hypersensitivity is still a critical clinical issue, and the underlying molecular mechanisms have remained unresolved to date. In this study, we found that repeated cisplatin treatments remarkedly upregulated the P2Y12 expression in the spinal cord. Expression of P2Y12 was predominant in the microglia. Pharmacological inhibition of P2Y12 expression markedly attenuated the cisplatin-induced pain hypersensitivity. Meanwhile, blocking the P2Y12 signal also suppressed cisplatin-induced microglia hyperactivity. Furthermore, the microglia Src family kinase/p38 pathway is required for P2Y12-mediated cisplatin-induced pain hypersensitivity via the proinflammatory cytokine IL-18 production in the spinal cord. Blocking the P2Y12/IL-18 signaling pathway reversed cisplatin-induced pain hypersensitivity, as well as activation of N-methyl-D-aspartate receptor and subsequent Ca2+-dependent signals. Collectively, our data suggest that microglia P2Y12-SFK-p38 signaling contributes to cisplatin-induced pain hypersensitivity via IL-18-mediated central sensitization in the spinal, and P2Y12 could be a potential target for intervention to prevent chemotherapy-induced pain hypersensitivity. PERSPECTIVE: Our work identified that P2Y12/IL-18 played a critical role in cisplatin-induced pain hypersensitivity. This work suggests that P2Y12/IL-18 signaling may be a useful strategy for the treatment of chemotherapy-induced pain hypersensitivity.

Transcription factor ETS proto-oncogene 1 contributes to neuropathic pain by regulating histone deacetylase 1 in primary afferent neurons.

Nerve injury can induce aberrant changes in ion channels, enzymes, and cytokines/chemokines in the dorsal root ganglia (DRGs); these changes are due to or at least partly governed by transcription factors that contribute to the genesis of neuropathic pain. However, the involvement of transcription factors in neuropathic pain is poorly understood. In this study, we report that transcription factor (TF) ETS proto-oncogene 1 (ETS1) is required for the initiation and development of neuropathic pain. Sciatic nerve chronic constrictive injury (CCI, a clinical neuropathic pain model) increases ETS1 expression in the injured male mouse DRG. Blocking this upregulation alleviated CCI-induced mechanical allodynia and thermal hyperalgesia, with no apparent effect on locomotor function. Mimicking this upregulation results in the genesis of nociception hypersensitivity; mechanistically, nerve injury-induced ETS1 upregulation promotes the expression of histone deacetylase 1 (HDAC1, a key initiator of pain) via enhancing its binding activity to the HDAC1 promotor, leading to the elevation of spinal central sensitization, as evidenced by increased expression of p-ERK1/2 and GFAP in the dorsal spinal horn. It appears that the ETS1/HDAC1 axis in DRG may have a critical role in the development and maintenance of neuropathic pain, and ETS1 is a potential therapeutic target in neuropathic pain.

Possible association between viral infection and poor survival of the corneal graft after penetrating keratoplasty in patients with congenital corneal opacity: a cohort study.

BACKGROUND: Congenital corneal opacity (CCO) is a rare disorder. Penetrating keratoplasty (PK) is the main surgical option for CCO, but many factors affect graft survival. Therefore, this study aimed to perform a virological examination of CCO specimens after PK to explore the relationship between virological factors and graft survival after PK. METHODS: This prospective study included consecutive patients (<6 months of age) diagnosed with CCO and treated with PK at Beijing Tongren Hospital from August 2017 to January 2018. Next-generation sequencing was used to detect viral DNA in the CCO specimens. The survival of the primary graft was analysed using the Kaplan-Meier method. RESULTS: Overall, 24 eyes of 24 infants were treated with PK during the study period. The mean age at surgery was 4.8±1.1 months. Epstein-Barr virus DNA was detected in two specimens, varicella-zoster virus DNA in one specimen, herpes simplex virus DNA in three specimens and cytomegalovirus DNA in one specimen. In the virus-positive group, only one (14.3%) graft remained clear during follow-up. In contrast, in the virus-negative group (n=17), 13 (76.5%) grafts were still clear at the last follow-up. The mean survival of the grafts in the virus-positive group was significantly shorter than in the virus-negative group (11.0±9.8 months vs 27.1±7.7, p<0.001). CONCLUSION: The presence of viral DNA in CCO specimens might be associated with poor graft survival after PK.

The Protective Effects of KAT5 Inhibition on Ocular Inflammation by Mediating the PI3K/AKT Pathway in a Murine Model of Allergic Conjunctivitis.

Purpose: We aimed to explore the effect of lysine acetyltransferase KAT5 on allergic conjunctivitis (AC). Methods: The effect of KAT5 on inflammatory response during AC progression was analyzed in the experimental allergic conjunctivitis (EAC) mouse model. Results: The clinical score, permeability, total IgE, ovalbumin (OVA)-specific IgE, and IgG1/IgG2a were induced in the EAC mice, in which the overexpression of KAT5 could further enhance but KAT5 inhibitor NU9056 reduce the phenotypes. The eosinophilic infiltration was induced in EAC mice, in which the overexpression of KAT5 was able to further promote but NU9056 attenuate the phenotype. The expression of Eotaxin and RANTES and the inflammatory factors were upregulated in EAC mice and KAT5 overexpression increased, but NU9056 decreased the expression in the model. Significantly, the CD11c+ dendritic cells and CD4+ T cells infiltration in the conjunctiva was enhanced in EAC mice, whereas KAT5 overexpression induced but NU9056 suppressed the effect in the model. Mechanically, the phosphorylation of PI3K and Akt and the levels of histone H3 lysine 27 acetylation (H3K27ac) were enhanced in EAC mice, whereas the overexpression of KAT5 promoted and NU9056 repressed the phenotype in the mice. The enrichment of KAT5 and H3K27ac on PI3K promoter was increased in EAC mice, and the overexpression of KAT5 further enhanced the enrichment in the mice. Significantly, we observed similar results in the KAT5 knockout mice as well. Moreover, PI3K/AKT signaling inhibitor LY294002 reversed KAT5 overexpression-mediated phenotypes and inflammatory response after induction AC in vivo. Conclusions: Therefore we concluded that KAT5 inhibition protected against ocular inflammation by mediating the PI3K/AKT pathway in EAC mouse model.

[Clinical study of type â ¢ prostatitis treated with "warming and promoting technique" of acupuncture combined with extracorporeal shock wave].

OBJECTIVE: To observe the clinical curative effect of wentongzhenfa (warming and promoting technique of acupuncture) combined with extracorporeal shock wave in treatment of type Ⅲ prostatitis. METHODS: A total of 96 patients with type Ⅲ prostatitis were randomly divided into an observation group (48 cases) and a control group (48 cases). In the control group, the extracorporeal shock wave was combined with even-needling technique of acupuncture at Guanyuan (CV4), Mingmen (GV4), Zhongji (CV3), Zusanli (ST36), etc. In the observation group, the extracorporeal shock wave was combined with "warming and promoting technique" of acupuncture at the same acupoints as the control group. The treatment lasted 30 min each time, once daily in either group. There were 2 days of interval after consecutive treatment for 5 days. Totally, the duration of treatment was 1 month in two groups. The clinical curative effect was assessed after treatment. Before and after treatment, the changes in the concentrations of tumor nerosis factor-α (TNF-α), interleukin-1ß(IL-1ß) and IL-6 in prostatic fluid were determined; and the symptoms were scored, i.e. frequent, urgent and burning painful urine, difficulty in urination, dribbling urine, distending pain in perineum, bitter taste and dry mouth, and scrotal dampness. The changes in the scores of National Institute of Health chronic prostatitis symptom index (NIH-CPSI), international index of erectile function (IIEF), visual analogue scale (VAS) were evaluated befroe and after treatment. Successively, before treatment, after treatment, as well as 1 and 3 months after treatment, the quality of life was evaluated by Karnofsky in the patients of two groups. RESULTS: After treatment, the total effective rate was 89.6% (43/48) in the observation group, higher than 70.8% (34/48) in the control group (P<0.05). Compared with those before treatment, the concentrations of TNF-α and IL-1ß in the prostatic fluid were all decreased (P<0.05) and the concentration of IL-6 was increased (P<0.05), the scores of symptoms, NIH-CPSI, IIEF and VAS were all reduced (P<0.05) in two groups. The changes of the above indexes were more obvious in the observation group than those in the control group (P<0.05). After treatment and 1 and 3 months after treatment, Karnofsky scores all increased (P<0.05) in two groups，and the increases were more significant in the observation group (P<0.05). CONCLUSION: "Warming and promoting technique" of acupuncture combined with extracorporeal shock wave promotes the elimination of local inflammatory factors, relieves clinical symptoms, improves the quality of life, as well as has a satisfactory short-term and medium-term curative effect on type Ⅲ prostatitis.

Targeted delivery of maytansine to liver cancer cells via galactose-modified supramolecular two-dimensional glycomaterial.

A two-dimensional (2D) glycomaterial for targeted delivery of maytansine to liver cancer cells was developed. Host-guest interaction between a galactosyl dye and human serum albumin (HSA) produces supramolecular galactoside-HSA conjugates, which are then used to coat 2D MoS2. The 2D glycomaterial was shown to be capable of the targeted delivery of maytansine to a liver cancer cell line that highly expresses a galactose receptor, resulting in greater cytotoxicity than maytansine alone.

Germinal peptide eye drop promotes corneal epithelial and stromal defect healing in rabbit model.

OBJECTIVE: Corneal defect is a common disease in ophthalmology caused by trauma, inflammation, drug toxicity, or surgery. To investigate the effect of germinal peptide eye drop on corneal epithelial and stromal defects after lamellar keratectomy in rabbit model. METHODS: Eighty-five male New Zealand white rabbits were divided into five groups: Germinal Peptide eye drop at three different concentration groups, normal saline (negative control group), recombinant human epidermal growth factor (rh-EGF) eye drop (positive control group). Corneal epithelial and stromal defects of around 150-200 µm in depth were created with an 8 mm diameter trephine in the center of the right eyes of all animals. RESULTS: Germinal peptide eye drop with the concentration of 0.001%, 0.002%, and 0.004% and rh-EGF eye drop were more effective in promoting healing, reducing opacity, and edema during the process of corneal epithelial and stromal defect regeneration compared with 0.9% normal saline. No significant difference was observed among the three different doses of germinal peptide eye drop. Compared with the saline control group, the structures of the regenerated corneas were more orderly and less inflammatory cell infiltration was observed in the germinal peptide eye drop groups and the rh-EGF eye drop group. CONCLUSION: Germinal peptide eye drop (0.001%, 0.002%, and 0.004%) can significantly stimulate the regeneration of corneal epithelia and stroma and reduce corneal opacity and edema. Dose dependency was not observed in the current study.

α-Hederin inhibits the platelet activating factor-induced metastasis of HCC cells through disruption of PAF/PTAFR axis cascaded STAT3/MMP-2 expression.

Metastasis remains a crucial obstacle to the clinical treatment of hepatocellular carcinoma (HCC). Investigating the potential anti-tumor compounds from medicinal herb against HCC metastasis is of particular interest. As a triterpenoid saponin, α-Hederin has been reported to exhibit cytotoxicity for diverse cancer cell lines by inducing mitochondrial related apoptosis or autophagic cell death. Nevertheless, little is known about the inhibitory effect of α-Hederin on the metastasis of HCC and its underlying mechanisms. Here, we integrated well-established target prediction webtool and molecular docking methods to predict the potential targets for α-Hederin, and finally focused on PTAFR, the receptor for platelet-activating factor (PAF). Activation of PAF/PTAFR pathways has been reported to be contribution to the initiation and progression of cancer. We showed for the first time that non-cytotoxic concentration of α-Hederin inhibited cell migration and invasion induced by PAF in HCC cells, as well as lung metastasis in vivo. Moreover, we demonstrated α-Hederin reduced the PAF-induced matrix metalloproteinase-2 expression through inhibiting the activation of STAT3 in PAF stimulated HCC cells. These findings suggest that α-Hederin functions as a prospective inhibitor of PTAFR and may be utilized as an optional candidate for treatment of HCC.

Local Immunosuppression in Wuzhishan Pig to Rhesus Monkey Descemet's Stripping Automated Endothelial Keratoplasty: An Innovative Method to Promote the Survival of Xenografts.

INTRODUCTION: Corneal xenotransplantation is an effective solution for human corneal shortage. We investigated the feasibility and efficacy of different postoperative protocols on xeno-Descemet's stripping automated endothelial keratoplasty (DSAEK) grafts. METHODS: Thirty rhesus monkeys were randomly divided into three groups: control group (C) and only Descemet's membrane (DM) stripping, DSAEK 1 (D1) and DSAEK 2 (D2) groups, DM stripping followed by endothelial keratoplasty. Betamethasone 3.5 mg was subconjunctivally injected in groups control and D1 postoperatively, whereas rhesus monkeys in group D2 received topical 0.1% tacrolimus and topical steroids. All groups were evaluated by slit lamp, anterior segment optical coherence tomography, and laser scanning confocal microscopy for at least 9 months. RESULTS: Twenty-four monkeys met the inclusion criteria. Nine months after the DSAEK surgery, most corneas were transparent. Graft rejection was observed in 25% and 28.57% of the cases in group D1 and group D2 (p > 0.05), respectively. Corneal endothelium densities in DSAEK groups were 2,715.83 ± 516.20/mm2 (D1) and 2,220.00 ± 565.13/mm2 (D2) (p > 0.05). CONCLUSIONS: Xenogeneic corneal endothelial grafts can survive and function in rhesus monkey eyes for a prolonged period of time with subconjunctival steroid or topical tacrolimus and steroid treatment. Furthermore, topical drugs are more suitable for clinical use.

Spliceostatin C, a component of a microbial bioherbicide, is a potent phytotoxin that inhibits the spliceosome.

Spliceostatin C (SPC) is a component of a bioherbicide isolated from the soil bacterium Burkholderia rinojensis. The chemical structure of SPC closely resembles spliceostatin A (SPA) which was characterized as an anticancer agent and splicing inhibitor. SPC inhibited the growth of Arabidopsis thaliana seedlings with an IC50 value of 2.2 µM. The seedlings exposed to SPC displayed a significant response with decreased root length and number and inhibition of gravitropism. Reverse transcriptase semi-quantitative PCR (RT-sqPCR) analyses of 19 selected genes demonstrated the active impact of SPC on the quality and quantity of transcripts that underwent intron rearrangements as well as up or down expression upon exposure to SPC. Qualitative and quantitative proteomic profiles identified 66 proteins that were significantly affected by SPC treatment. Further proteomics data analysis revealed that spliceostatin C induces hormone-related responses in Arabidopsis seedlings. In silico binding studies showed that SPC binds to a pocket between the SF3B3 and PF5A of the spliceosome.

Adenovirus-mediated RNA interference against herpes simplex virus infection in vitro.

INTRODUCTION: Herpetic keratitis caused by the herpes simplex virus (HSV) is the most common form of ocular herpes that causes corneal blindness. Although treatments for herpes keratitis have improved in recent years. there is still considerable room for new treatments against viral infection that shows great promise. The aim of the study was to evaluate the effect of RNA interference on HSV Type 1 (HSV1) infection in vitro, first prophylactically then therapeutically. MATERIAL AND METHODS: The highly conserved glycoproteins D (gD) and E (gE) were chosen as targets for this study. Different small interfering RNA (siRNA) duplexes that target gD and gE were designed and chemically synthesized. The recombinant adenovirus type 5 was developed and used as the vehicle with which we delivered the siRNA into the Vero cells infected with the HSV1 KOS strain. Evaluation of the efficacy of siRNA-mediated inhibition was performed either before virus inoculation (prophylactically) or after virus inoculation at the first appearance of lesions (therapeutically). The expression of messenger RNA encoding gD and gE was detected using a real-time polymerase chain reaction (qPCR). We analyzed HSV replication in Vero cells, cytotoxicity of HSV, and cell viability. RESULTS: When used prophylactically, the siRNA-targeting gD and gE created a more marked decrease in viral titer than when used therapeutically. The transfection of cells with recombinant adenovirus containing the siRNA expression cassette was associated with very low cytotoxicity. CONCLUSIONS: Adenovirus-mediated siRNA-targeting gD and gE genes effectively inhibit the replication of the HSV in Vero cells. In addition, these findings indicate that the prophylactic use of siRNA is far more effective at inhibiting HSV replication than the therapeutic use.

Methyltransferase-like 3 contributes to inflammatory pain by targeting TET1 in YTHDF2-dependent manner.

ABSTRACT: The methyltransferase-like 3 (Mettl3) is a key component of the large N6-adenosine-methyltransferase complex in mammalian responsible for RNA N6-methyladenosine (m6A) modification, which plays an important role in gene post-transcription modulation. Although RNA m6A is enriched in mammalian neurons, its regulatory function in nociceptive information processing remains elusive. Here, we reported that Complete Freund's Adjuvant (CFA)-induced inflammatory pain significantly decreased global m6A level and m6A writer Mettl3 in the spinal cord. Mimicking this decease by knocking down or conditionally deleting spinal Mettl3 elevated the levels of m6A in ten-eleven translocation methylcytosine dioxygenases 1 (Tet1) mRNA and TET1 protein in the spinal cord, leading to production of pain hypersensitivity. By contrast, overexpressing Mettl3 reversed a loss of m6A in Tet1 mRNA and blocked the CFA-induced increase of TET1 in the spinal cord, resulting in the attenuation of pain behavior. Furthermore, the decreased level of spinal YT521-B homology domain family protein 2 (YTHDF2), an RNA m6A reader, stabilized upregulation of spinal TET1 because of the reduction of Tet1 mRNA decay by the binding to m6A in Tet1 mRNA in the spinal cord after CFA. This study reveals a novel mechanism for downregulated spinal cord METTL3 coordinating with YTHDF2 contributes to the modulation of inflammatory pain through stabilizing upregulation of TET1 in spinal neurons.