Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions.

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1V and TET1HxD rescue most hypermethylation of Tet1-/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning.

γH2A/γH2AX Mediates DNA Damage-Specific Control of Checkpoint Signaling in Saccharomyces cerevisiae.

DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In Saccharomyces cerevisiae, phosphorylation of histone H2A (γH2A, equivalent to γH2AX in mammals) is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that γH2A negatively regulates the G2/M checkpoint in response to the genotoxin camptothecin, which is a DNA topoisomerase I poison. γH2A also suppresses DDC signaling induced by the DNA alkylating agent methyl methanesulfonate. These results differ from prior findings, which demonstrate positive or no roles of γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, which suggest that γH2A has DNA damage-specific effects on DDC signaling. We also find evidence supporting the notion that γH2A regulates DDC signaling by mediating the competitive recruitment of the DDC mediator Rad9 and the DNA repair factor Rtt107 to DNA lesions. We propose that γH2A/γH2AX serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

Clinical features and 18F-FDG PET/CT for distinguishing of malignant lymphoma from inflammatory lymphadenopathy in HIV-infected patients.

BACKGROUND: It is vital to distinguish between inflammatory and malignant lymphadenopathy in human immunodeficiency virus (HIV) infected individuals. The purpose of our study was to differentiate the variations in the clinical characteristics of HIV patients, and apply 18F-FDG PET/CT parameters for distinguishing of malignant lymphoma and inflammatory lymphadenopathy in such patients. METHODS: This retrospective cross-sectional study included 59 consecutive HIV-infected patients who underwent whole-body 18F-FDG PET/CT. Of these patients, 37 had biopsy-proven HIV-associated lymphoma, and 22 with HIV-associated inflammatory lymphadenopathy were used as controls. The determined parameters were the maximum of standard uptake value (SUVmax), SUVmax of only lymph nodes (SUVLN), the most FDG-avid lesion-to-liver SUVmax ratio (SURmax), laboratory examinations and demographics. The optimal cut-off of 18F-FDG PET/CT value was analyzed by receiver operating characteristic curve (ROC). RESULTS: Considering the clinical records, the Karnofsky Performance Status (KPS) scores in patients with inflammatory lymphadenopathy were obviously higher than those in patients with malignant lymphoma (P = 0.015), whereas lymphocyte counts and lactate dehydrogenase (LDH) were obviously lower (P = 0.014 and 0.010, respectively). For the 18F-FDG PET/CT imaging, extra-lymphatic lesions, especially digestive tract and Waldeyer's ring, occurred more frequently in malignant lymphoma than inflammatory lymphadenopathy. Furthermore, the SURmax and SUVLN in malignant lymphoma were markedly higher than those in inflammatory lymphadenopathy (P = 0.000 and 0.000, respectively). The cut-off point of 3.1 for SURmax had higher specificity (91.9%) and relatively reasonable sensitivity (68.2%) and the cut-off point of 8.0 for the SUVLN had high specificity (89.2%) and relatively reasonable sensitivity (63.6%). CONCLUSION: Our study identified the distinctive characteristics of the clinical manifestations, the SURmax, SUVLN and detectability of extra-lymphatic lesions on 18F-FDG PET, and thus provides a new basis for distinguishing of malignant lymphoma from inflammatory lymphadenopathy in HIV-infected patients.

Short-term effects of intravitreal Conbercept injection combined with laser photocoagulation on macular edema secondary to ischemic retinal vein occlusion.

AIM: To observe changes in the best-corrected visual acuity (BCVA), central macular thickness (CMT), and central choroidal thickness (CCT) of patients with macular edema (ME) secondary to ischemic retinal vein occlusion (iRVO) following intravitreal Conbercept injection. METHODS: This retrospective study included 33 eyes from 33 patients who received intravitreal injections of Conbercept for ME secondary to iRVO. Treatments were performed on a 3+pro re nata (3+PRN) basis. All of the patients were examined by fundus fluorescein angiography and spectral domain optical coherence tomography at the first visit. Laser photocoagulation was performed in the nonperfusion area of the retina of all eyes after the first injection. BCVA, CMT, and CCT were observed before and after 6mo of treatment. The number of injections necessary to achieve improved vision was also noted. RESULTS: Following Conbercept treatment, the mean BCVA significantly improved from 0.81±0.39 at baseline to 0.41±0.25 and 0.43±0.29 logMAR in the third and sixth months, respectively (both P=0.000). The CMT of the patients at baseline was 556.75±98.57 µm; 304.78±68.53 and 306.85±76.77 µm 3 and 6mo after treatment, respectively (both P=0.000 vs baseline). The CCTs of the patients at baseline, 3 and 6mo after treatment were 304.63±57.83, 271.31±45.53, and 272.29±39.93 µm, respectively (P=0.026 and 0.035 vs baseline). No severe adverse event relevant to the therapy was noted, and the average number of injections delivered was 3.35. CONCLUSION: Intravitreal Conbercept injection combined with laser photocoagulation appears to be a safe and effective treatment for ME secondary to iRVO in the short-term.

Cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis on murine photoreceptor cells.

AIM: To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis (EAU) as well as their secreted interferon (IFN)-γ and interleukin (IL)-17A on murine photoreceptor (661W) cells. METHODS: An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein (IRBP) emulsion supplemented with complete Freund's adjuvant (CFA) and Mycobacterium tuberculosis (TB). On day 12 after induction of EAU, specific T cells from spleen and lymph node tissues were isolated and cultured for 4d and the levels of IFN-γ and IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). T cells and their supernatants were added to 661W cells to observe the alteration of cell morphology; IFN-γ and IL-17A were separately added to 661W cells to observe the effect of IFN-γ and IL-17A on cell proliferation. RESULTS: The levels of IFN-γ and IL-17A in the T cell supernatants were 1568.64±38.79 pg/mL and 1456.57±46.98 pg/mL, respectively. The supernatants apparently inhibited 661W cell proliferation (P<0.05). T cells could also attach to the surface of 661W cells, and IFN-γ showed a more serious cytotoxic effect on 661W cells than IL-17A, inhibiting cell proliferation (P<0.01). CONCLUSION: IFN-γ and IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation, and IFN-γ shows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.

Diode laser transscleral cyclophotocoagulation for uveitis-glaucoma-hyphema syndrome: A case report.

RATIONALE: To report a case of diode laser transscleral cyclophotocoagulation (DLTSC) for uveitis-glaucoma-hyphema syndrome (UGH). PATIENT CONCERNS: The patient developed UGH on the right eye (OD) after vitrectomy and intraocular lens (IOL) implantation. DIAGNOSES: Best corrected visual acuity (BCVA) was HM/50 cm, intraocular pressure (IOP) was 51.3 mm Hg on the OD. He was found to have 3+ anterior chamber cells. A B-scan ultrasound showed vitreous opacity. Ultrasound biomicroscopy (UBM) showed the chafing between the IOL and the posterior surface of the iris. Thus, he was diagnosed as UGH on the OD. INTERVENTIONS: The patient was worried about the complications for removal of the IOL, a DLTSC approach was performed. OUTCOMES: BCVA was 20/40 on the OD, IOP was 12 mm Hg on the OD. There were no anterior chamber inflammation and no vitreous opacity. UBM showed there was no contact between IOL and the posterior surface of the iris, the fundus of the eye was clearly visible. LESSONS: UGH syndrome is a severe complication of cataract extraction. IOL extraction has been the traditional approach to treatment. DLTSC can be an option when the IOL is slightly tilted.

Central retinal venous occlusion in a child with hyperhomocysteinemia: A case report.

RATIONALE: To report a rare case of 8-year-old girl patient with central retinal venous occlusion (CRVO) with hyperhomocysteinemia. PATIENT CONCERNS: The patient had a 2-year history on painless visual loss in the left eye. DIAGNOSES: All examination results were within normal limits except plasma homocysteine (HCY). Fluorescein angiography (FA) confirmed peripheral capillary non-perfusion (CNP) in the left eye, and OCT showed macular edema. The girl patient was diagnosed as CRVO. INTERVENTIONS: Based on all of the test results, laser photocoagulation was performed at peripheral capillary non-perfusion (NP). Ranibizumab was injected into virtreous cavity to reduce the macular edema. Oral folic acid, vitamin B12, and vitamin B6 were performed to the girl. OUTCOMES: After 13 months, the girl visual acuity recovered to 20/100 in the left eye. LESSONS: All eye examinations should be performed in young patients, and they should undergo treatments immediately after is diagnosed as CRVO.

Antimicrobial effect and mechanism of cinnamon oil and gamma radiation on Shewanella putrefaciens.

The aims of this study were to observe the antimicrobial effect and mechanism of cinnamon oil combined with gamma radiation on Shewanella putrefaciens. Gamma radiation increased the antimicrobial activity of cinnamon oil, and the relative radiation sensitivity of gamma radiation on S. putrefaciens was increased by cinnamon oil. Gamma radiation significantly increased the changes of bacterial morphology, intra-adenosine 5'-triphosphate (intra-ATP) and extra-ATP concentrations and pHin value of S. putrefaciens treated cinnamon oil. Although, gamma radiation used alone didn't damage the bacterial morphology and ATP concentrations significantly. Gamma radiation assisted cinnamon oil to damage the cell permeability and integrity of S. putrefaciens, thus the combination of cinnamon oil and gamma radiation showed a better antimicrobial activity than used alone.

Unusual synchronous liver and brain abscesses infected by rare Aerococcus viridians in a patient with pulmonary arteriovenous malformations on FDG PET/CT: A case report and literature review.

RATIONALE: Pulmonary arteriovenous malformations (PAVMs) complicated with multiple organ abscesses is an uncommon manifestation. Because of the low incidence of the disease, F-18 fluorodeoxyglucose positron emission tomography with computed tomography (F-FDG PET/CT) imaging studies for PAVMs complicated with multiple organ abscesses are scarce. PATIENT CONCERNS: We report a case of a 54-year-old man presenting with PAVMs complicated with synchronous multiple organ abscesses founded by F-FDG PET/CT. F-FDG PET/CT revealed tortuous stripes and mass opacities with no significant FDG uptake in the left upper lung lobe. However, hypermetabolic lesions located in the anterior inferior segment of right hepatic lobe [with maximum standardized uptake value (SUVmax) of 10.7], and in the right basal ganglia with SUVmax of 14.1 were found by F-FDG PET/CT. DIAGNOSES: A diagnosis of synchronous liver and brain abscesses infected by rare Aerococcus viridans was determined by tissue culture. INTERVENTIONS: Vancomycin was provided intravenously, and oral linezolidate tablets were prescribed for anti-inflammatory treatment for 1 month. Liver and head magnetic resonance imaging was performed during the follow-up. OUTCOMES: The lesion in the right basal ganglia was reduced, and the lesion in the right liver had disappeared, indicating the lesions were abscesses. LESSONS: The present case indicated that the possibility of abscesses should be considered with patients with PAVMs, and whole-body F-FDG PET/CT is suggested to identify possible accompanying abscesses in multiple organs for PAVMs patients.

Synthesis and cytotoxic activity of novel hexahydropyrrolo[2,3-b]indole imidazolium salts.

A series of novel hexahydropyrrolo[2,3-b]indole-1H-imidazolium salts were synthesized and evaluated in vitro against a panel of human tumor cell lines. The results suggest that the 5,6-dimethyl-benzimidazole ring, and substitution of the imidazolyl-3-position with a 2-bromobenzyl or 2-naphthylmethyl group, were important for the cytotoxic activity. Notably, Compound 43, bearing a 2-bromobenzyl substituent at position-3 of 5,6-dimethyl-benzimidazole, was found to possess the most potent derivative against five human tumor cell lines with IC50 values below 2.68µM and more selective towards SMMC-7721, A549 and SW480 cell lines. Compounds 25 and 39 were more selective to HL-60 and MCF-7 cell lines with IC50 values of 0.47 and 1.46µM.

Characterization of microRNA expression profiling in peripheral blood lymphocytes in rats with experimental autoimmune uveitis.

OBJECTIVE AND DESIGN: We aimed to investigate the alterations of microRNA (miRNA) genomics in peripheral blood lymphocytes in experimental autoimmune uveitis (EAU) rats versus control samples. MATERIALS/METHODS: Six Lewis rats received interphotoreceptor retinoid-binding protein (IRBP) emulsion to induce EAU. On day 12, peripheral blood lymphocytes were isolated, and total RNAs were extracted. Using microarray analysis, we analyzed the aberrant miRNAs, validated the relevant expression of differentially expressed miRNAs, and predicted the possible miRNA targets and signaling pathways. RESULTS: The results indicated that 36 miRNAs were upregulated and 31 miRNAs were downregulated in EAU rats versus normal samples. Real-time quantitative PCR substantiated a high degree of confidence for the differentially expressed miRNAs, and miRNA analyses showed the differentially expressed miRNA targets were involved not only in the multicellular organismal process and developmental process, but also in T cell receptor signaling pathway, B cell receptor signaling pathway and so on. CONCLUSIONS: Our findings show that the differentially expressed miRNAs in EAU rats were closely associated with immune signaling pathways and may be applied in early prevention, prognosis and possible therapy in uveitis, indicating that miRNAs play an important role in the development of uveitis.

Pathogenesis of innate immunity and adaptive immunity in the mouse model of experimental autoimmune uveitis.

Experimental autoimmune uveitis, a well-established model for human uveitis, is similar to human uveitis in many pathological features. Studies concerning the mechanisms of experimental autoimmune uveitis would cast a light on the pathogenesis of human uveitis as well as the search for more effective therapeutic agents. The cellular components of innate immunity include natural killer cells, gamma delta T lymphocytes, antigen-presenting dendritic cells, phagocytic macrophages, and granulocytes. It is believed that T cells are central in the generation of human uveitis. It has already become clear that CD4(+) effecter cells that predominantly produce interleukin-17 (the so-called Th17 cells) may play an important role in uveitis. In addition, the occurrence and recurrence of uveitis depends on a complex interplay between the elements of innate and adaptive immunity.

Proteomic analysis of rat plasma with experimental autoimmune uveitis based on label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Uveitis is a severe autoimmune eye disease that can cause intraocular inflammation even lead to severe vision loss, and the occurrence of uveitis can be closely associated with abnormal expression of proteins. However, the abnormally expressed proteins involved in uveitis are not well identified. Using liquid chromatography-tandem mass spectrometry technique, we examined the alterations in proteomic expression profiling in rat plasma specimens related to experimental autoimmune uveitis (EAU) versus normal samples. In addition, the experimental verification was further performed using enzyme-linked immunosorbent assay (ELISA) for abnormally expressed proteins in EAU rat plasma. The results indicate that 62 proteins were upregulated and 106 proteins were downregulated in plasma from EAU rats compared with those in saline-treated samples. In the meantime, we observed that the plasma level of complement component 3 in EAU rats was upregulated versus saline-treated rats (from 92.32µg/mL to 168.92µg/mL), whereas the level of interleukin-1 receptor accessory protein was downregulated (from 1120.97pg/mL to 798.39pg/mL), and these results were highly in agreement with those of mass spectrometry determination. Taken together, our results indicate that liquid chromatography-tandem mass spectrometry analysis possesses a good resolution for peptides in plasma, and the findings will provide the baseline plasma dataset for EAU rats and the relevant information can contribute to future studies on the understanding the mechanism of uveitis.

Treatment of Valsalva retinopathy.

PURPOSE: To report a case of Valsalva retinopathy treated by neodymium-doped yttrium aluminum garnet (Nd:YAG) laser and discuss the previously reported treatments for Valsalva retinopathy. CASE REPORT: A 35-year-old-woman experienced a sudden painless visual loss in her left eye after lifting heavy bags of manure on the farm. The patient was diagnosed as having Valsalva retinopathy. The examination revealed a macular preretinal hemorrhage. An Nd:YAG laser was used on the patient after conservative management afforded no resolution. After treatment, visual acuity improved. CONCLUSIONS: Valsalva retinopathy can be treated by conservative management, Nd:YAG laser, and vitrectomy, but each treatment must be evaluated in regard to the duration, the location, and the amount of the hemorrhage.

The proliferation of malignant melanoma cells could be inhibited by ranibizumab via antagonizing VEGF through VEGFR1.

PURPOSE: Angiogenesis is an important mediator in tumor progression. Vascular endothelial growth factor (VEGF) is one of the major cytokines that can influence angiogenesis. However, the potential mechanism of tumor growth inhibition through anti-VEGF agents is still unclear. This study was performed to examine whether ranibizumab could inhibit malignant melanoma growth in vitro and to determine the safety of ranibizumab on human adult retinal pigment epithelium cell line (ARPE-19 cells). METHODS: Malignant melanoma cells obtained from a clinic were cultured in vitro. VEGF concentrations secreted by malignant melanoma cells and the ARPE-19 cells were examined by enzyme-linked immunosorbent assay (ELISA). The two kinds of cells were both treated with VEGF and its antagonist, ranibizumab. The dynamic changes of the two types of cells were monitored by real-time cell electronic sensing (RT-CES) assay. The effect of ranibizumab on both types of cells was verified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The expression of VEGF receptor 1 (VEGFR1) RNA in uveal melanoma was further investigated through the PCR technique. RESULTS: The levels of VEGF secreted by malignant melanoma cells were much higher than those of ARPE-19 cells, and were markedly decreased in the action of 0.1 mg/ml ranibizumab. However, there was no obvious reduction of VEGF in the presence of ranibizumab for ARPE-19 (p>0.05). Meanwhile, RT-CES showed that the viability of malignant melanoma cells increased greatly in the presence of VEGF. When VEGF was 20 ng/ml, viability of the malignant melanoma cells increased by 40% compared with the negative control. There was no evident effect on proliferation of ARPE-19 (p>0.05). Furthermore, the growth of malignant melanoma cells was obviously inhibited after ranibizumab intervention. When ranibizumab was administered at 0.25 mg/ml, the survival rate of the malignant melanoma cells decreased to 57.5%. Nevertheless, low-dose exposure to ranibizumab had only a slight effect on the growth of ARPE-19, and PCR result demonstrated that VEGFR1 plays a role in this tumor tissue rather than VEGFR2. CONCLUSIONS: Ranibizumab can selectively inhibit malignant melanoma cell proliferation by decreasing the expression of VEGF; the possible mechanism of the inhibitory effect may involve VEGFR1 antagonism.

Effective target size for the induction of bystander effects in medium transfer experiments.

Although radiation-induced bystander effects are frequently observed biological phenomena, the mechanism for these effects has not been fully determined. The target-hit theory and related concepts from microdosimetry provide a convenient formalism to help identify the nature of the targets responsible for initiating the emission of diffusible factors in medium transfer experiments. We used the microdosimetric models proposed by Stewart et al. (Radiat. Res. 165, 460-469, 2006) to analyze the results of published medium transfer experiments for gamma-ray doses in the range of 0.04 mGy to 5 Gy. The analysis suggests that the effective size of the target responsible for initiating signal emission in HPV-G human keratinocyte donor cells is approximately 2 microm.

A dose threshold for a medium transfer bystander effect for a human skin cell line.

The existence of radiation-induced bystander effects mediated by diffusible factors is now accepted, but the mechanisms and precise behavior at low doses remain unclear. We exposed cells to gamma-ray doses in the range 0.04 mGy-5 Gy, harvested the culture medium, and transferred it to unirradiated reporter cells. Calcium fluxes and clonogenic survival were measured in the recipients. We show evidence for a dose threshold around 2 mGy for the human skin cell line used with a suggestion of increased survival below that dose. Similar experiments using direct gamma irradiation showed no reduction in survival until the dose exceeded 7 mGy. Preliminary data for neutrons where the gamma-ray dose was kept below the bystander threshold do not show a significant bystander effect in the dose range 1-33 mGy. A lack of a bystander response with neutrons occurred at around 1 Gy, where significant cell killing from direct irradiation was observed. The result may have implications for understanding the role of bystander effects at low doses.