Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity

Recent clinical studies have shown that mutation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in cancer cells may be associated with immunosuppressive tumor microenvironment (TME) and poor response to immune checkpoint blockade (ICB) therapy. Therefore, efficiently restoring PTEN gene expression in cancer cells is critical to improving the responding rate to ICB therapy. Here, we screened an adeno-associated virus (AAV) capsid for efficient PTEN gene delivery into B16F10 tumor cells. We demonstrated that intratumorally injected AAV6-PTEN successfully restored the tumor cell PTEN gene expression and effectively inhibited tumor progression by inducing tumor cell immunogenic cell death (ICD) and increasing immune cell infiltration. Moreover, we developed an anti-PD-1 loaded phospholipid-based phase separation gel (PPSG), which formed an in situ depot and sustainably release anti-PD-1 drugs within 42 days in vivo. In order to effectively inhibit the recurrence of melanoma, we further applied a triple therapy based on AAV6-PTEN, PPSG@anti-PD-1 and CpG, and showed that this triple therapy strategy enhanced the synergistic antitumor immune effect and also induced robust immune memory, which completely rejected tumor recurrence. We anticipate that this triple therapy could be used as a new tumor combination therapy with stronger immune activation capacity and tumor inhibition efficacy.

Progress in research on B class CpG oligodeoxynucleotides in bacterial vaccines / 中国生物制品学杂志

@#CpG oligonucleotide(CpG ODN)is a synthetic oligodeoxynucleotide containing non-methylated CpG,which has broad application prospects in disease prevention and clinical treatment. Among them,B class CpG ODN is widely used in vaccine research because of its strong immune stimulation and some motifs have entered the clinical research stage. At the same time,the problem of bacterial resistance in clinical treatment has become increasingly serious,and the development of bacterial vaccine with B class CpG ODN as adjuvant has become a research hotspot. This paper reviewed the current research status and related progress of the existing B class CpG ODN 1826,2006,2007 and 1668 motifs in bacterial vaccines,with a view to providing a reference for the subsequent development and application of bacterial vaccines.

Progress in research on novel BC adjuvant system / 中国生物制品学杂志

@#Vaccines with novel adjuvants have been listed abroad,while in China,except for aluminum adjuvants widely used in vaccine research and production,few other novel adjuvants have been successfully listed. This paper briefly summarized the source,development history,research progress on biological activity and immune mechanism as well as safety evaluation of the novel BC adjuvant system with independent intellectual property right which has been applied to the vaccine in clinical research stage,so as to provide theoretical support for selection of the adjuvant in the development of novel vaccine.

UHRF1/DNMT1-MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

Evaluation of inhibitory effect of tumor vaccine in colon carcinoma model mice / 中国生物制品学杂志

Objective@#To evaluate the inhibitory effect of tumor vaccines in colon carcinoma model mice.@*Methods@# Mouse bone marrow⁃derived dendritic cells（BMDCs）were stimulated by using CpG β⁃glucan nanoparticles（CNP）in vitro. The BMDCs were divided into PBS group，NP group（without CpG nanoparticles），Lysate group（MC38 cell lysate）and CpG group（CpG1826），which were determined for the expression of marker molecules on the surface by flow cytometry and for the contents of interleukin⁃6（IL⁃6）and IL⁃12p40 in the culture supernatant by ELISA. The tumor lysate nano⁃vaccine was pre⁃ pared by mixing 50 mg／mL tumor lysate（MC38 cell lysate）with 200 mg／mL CNP in a volume ratio of 1∶1，with which mice were subcutaneously immunized as Vaccine group. Vaccine group，PBS group，CNP group and Lysate group were im⁃ munized once a week，for three times in total. Mice were subcutaneously inoculated with MC38 cells，2 × 105 cells for each， in the right lower limb 1 h after the last immunization，and measured for tumor volume once every three days to plot the tumor growth curve. The ratios of CD3+ CD4+ T and CD3+ CD8+ T cells in the blood were analyzed by flow cytometry and the levels of tumor necrosis factor⁃α（TNF⁃α）and interferon γ（IFNγ）in the blood and spleen of mice were determined by ELISA.@*Results@# CNP effectively increased the expression of CD11c+ CD80+，CD11c+ CD86+，CD11c+ MHC⁃Ⅱ+ and the secretion of IL⁃6 and IL⁃12p40 in BMDCs in vitro，which were significantly higher than those in other 4 groups（t = 4. 3 ~ 46. 2，each P < 0. 05）. Compared with that of the other three groups，the tumor volume of mice in Vaccine group decreased significantly（t =2.6~3.4，eachP <0. 05）；TherewasnosignificantdifferenceinCD3+ CD8+ TandCD3+ CD8+ Tcellratios（t = 0.5~ 1. 9，each P > 0. 05）；The content of IFNγ in blood increased significantly（t = 3. 8 ~ 4. 6，P < 0. 05），while thatof TNF⁃α showed no significant difference（t = 0. 4 ~ 2. 0，each P > 0. 05）；However，the contents of IFN γ and TNF⁃α in spleen increased significantly（t = 6. 3 ~ 13. 0，each P < 0. 001）.@*Conclusion@#The prepared nano⁃vaccine of tumor lysate improvedtheimmune level in mice and effectively inhibited the growth of colon carcinoma.

Crosstalk between CpG Methylation and Polymorphisms (CpG-SNPs) in the Promotor Region of DIO2 in Kashin-Beck Disease / 中国医学科学杂志(英文版)

Objective This study was designed to determine the methylation profile of four CpGs and the genotypes of two CpG-SNPs located in promoter region of DIO2 in patients with Kashin-Beck disease (KBD). We also analyzed the interaction between the CpGs methylations and CpG-SNPs. Methods Whole blood specimens were collected from 16 KBD patients and 16 healthy subjects. Four CpGs and two CpG-SNPs in the promoter regions of DIO2 were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The CpGs methylation levels were compared between samples from KBD patients and healthy subjects. The methylation levels were also analyzed in KBD patients with different CpG-SNP genotypes. Results The mRNA expression of DIO2 in whole blood of KBD patients was significnatly lower than in healthy controls (P <0.05). The methylation levels of DIO2-1_CpG_3 in KBD patients were significantly higher than those in healthy controls (P <0.05). The methylation levels of four CpGs were not significantly different between KBD patients and healthy controls. The methylation level of DIO2-1_CpG_3 in the promoter region of DIO2 in KBD patients with GA/AA genotype was significantly higher than that of KBD patients with GG genotype (P <0.05). Conclusion The methylation level of DIO2 increases in KBD patients. Similar trends exist in KBD carriers of variant genotypes of CpG-SNPs DIO2 rs955849187.

Effect of DC vaccine sensitized with CpG ODN combined with tumor antigen on murine melanoma / 药学学报

The potential application of dendritic cells (DC) sensitized with cytosine-phosphoric acid-guanine (CpG) oligodeoxynucleotide (ODN) and tumor antigen as a vaccine against murine melanoma was investigated with freshly isolated mouse bone marrow-derived dendritic cells. For the DC vaccine preparation, DC were sensitized with the B16 tumor antigen and CpG ODN was used to promote further maturation of the DC. The immunogenic activity of the vaccine was evaluated in vitro by determining the proliferation of T lymphocytes and the killing effect of cytotoxic T lymphocytes (CTL) on B16 tumor cells. The DC vaccine was injected intraperitoneally and tumor inhibition in mice bearing B16 xenografts was examined. All mice were cared for under an approved SIMM Institutional Animal Care and Use Committee (IACUC) protocol. In vitro, this DC vaccine promoted the proliferation of T lymphocytes and showed a potent killing effect on the target B16 cells. In vivo experiments showed that after treatment or pre-immunization both the tumor volume and weight were significantly decreased. The DC vaccine with CpG ODN and tumor antigen exhibited an inhibitory effect against melanoma, providing a potential method for melanoma cancer treatment.

Optimization of SARS-CoV-2 spike protein receptor binding domain expression in Pichia pastoris and evaluation of its immunogenicity / 中华微生物学和免疫学杂志

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Role of microRNA-132 and Mecp2 in methamphetamine dependence / 中国药理学通报

Aim To explore the roles of miRNA-132 and its related proteins(Mecp2, CREB)in the mechanism of methamphetamine(MA)-induced neurotoxicity and dependence.Methods The rats were intraperitioneally injected(ip)with MA(10 mg·kg-1·d-1)to establish methamphetamine dependence model with different dependent time courses of 1 week, 2 weeks, and 4 weeks respectively.The miRNA-132 and Mecp2 mRNA were detected by RT-qPCR, and the Mecp2, p-Mecp2, CREB and p-CREB proteins were detected by Western blot in the tissues of frontal cortex and hippocampus.Results In the frontal cortex, the miRNA-132 and Mecp2 mRNA were up-regulated in MA-dependent groups(P<0.05 and P<0.01), while the Mecp2 protein were down-regulated(P<0.01).MA could promote the phosphorylation of Mecp2 protein in the frontal cortex(P<0.01).In hippocampus, the miRNA-132 was down-regulated in the MA-dependent groups, but Mecp2 mRNA was up-regulated(P<0.05).Mecp2 protein increased in MA-dependent 1 week group(P<0.05), and then recovered with the prolonged time of MA dependence, then decreased in MA-dependent 4 weeks groups(P<0.05)in hippocampus.The phosphorylation level of Mecp2 was significantly decreased in the 1 week group(P<0.01), and then increased in the 2 weeks group(P<0.01)in hippocampus.Conclusions MA could induce an abnormal expression of miRNA-132 in the frontal cortex and hippocampus, and miRNA-132 might inhibit the translation of Mecp2 mRNA and induce the decrease expression of Mecp2 protein in the frontal cortex.But in hippocampus, miRNA-132 does not show the correlation with the Mecp2 expression trend of the frontal cortex.And miRNA-132 regulation does not depend on the expression of Mecp2 in hippocampus.

Síndrome de Rett: reporte de una nueva variante patogénica y revisión de la literatura a propósito de dos casos clínicos / Rett syndrome: report of a new pathogenic variant and review of the literature regarding two clinical cases

Resumen Introducción: El síndrome de Rett es un trastorno del neurodesarrollo con una frecuencia estimada de 1/10,000 recién nacidos vivos, el cual se presenta con un modelo de herencia ligado al cromosoma X. Las variantes patogénicas en el gen MECP2, el cual codifica para una proteína que participa en el desarrollo y la diferenciación del sistema nervioso central, causan este síndrome. El objetivo de este trabajo fue describir dos casos de síndrome de Rett, uno de ellos con una nueva variante del gen MECP2. Casos clínicos: El primer caso se trata de una paciente de 5 años con microcefalia y regresión del neurodesarrollo desde los 3 años. Clínicamente se diagnosticó de síndrome de Rett en estadio III. Se realizó la secuenciación del gen MECP2 y se identificó una variante probablemente patogénica en estado heterocigoto, c.606delC (p.Thr203Argfs*7), que no ha sido reportada previamente. El segundo caso es una paciente de 17 años, referida por discapacidad intelectual grave, que se encontró clínicamente en estadio IV. Se realizó la secuenciación de MECP2 y se identificó una variante patogénica [c.880C>T(p.Arg294*)] ya descrita previamente. Conclusiones: El diagnóstico clínico de síndrome de Rett se llevó a cabo con criterios establecidos. La confirmación diagnóstica fue mediante la secuenciación de MECP2. Para el correcto abordaje de los trastornos del neurodesarrollo es primordial conocer el fenotipo de síndrome de Rett, así como optar por el análisis molecular para la confirmación del diagnóstico. Los pacientes con síndrome de Rett requieren un seguimiento interdisciplinario para disminuir el impacto de las complicaciones.

Abstract Background: Rett syndrome is an X-linked neurodevelopmental disorder with an estimated frequency of 1/10,000 live births caused by hetereozygous pathogenic variants in the MECP2 gene, whose protein participates in the development and differentiation of the central nervous system. This study aimed to describe two cases with Rett syndrome diagnosis, one of them with a new variant of the MECP2 gene. Case reports: We first describe the case of a 5-year-old female with microcephaly and neurodevelopmental regression starting at 3 years old, clinically corresponding to stage III Rett syndrome. Sequencing of the MECP2 gene identified a heterozygous likely pathogenic variant [c.606delC (p.Thr203Argfs*7)] not reported previously. The second case is a 17-year-old female, referred due to severe intellectual disability, clinically found on stage IV. MECP2 sequencing was performed identifying a pathogenic variant previously described [c.880C> T (p.Arg294 *)]. Conclusions: Rett syndrome clinical diagnosis was carried out based on established criteria. MECP2 sequencing confirmed the diagnosis. For neurodevelopmental disorders approach, it is essential to know the phenotype of Rett syndrome and select the molecular tool for the diagnosis. Patients with Rett syndrome require interdisciplinary follow-up for reducing the impact of complications.

The Role of MeCP2 in Substance Addiction / 中国生物化学与分子生物学报

Substance addiction is considered to be a chronic recurrent encephalopathy. The neural adap-tation changes induced by addictive substances are partly mediated by epigenetic mechanism. The perma￣nent changes of gene expression in tissues or brain can be affected by DNA methylation, histone modifica-tion and chromatin remodeling, these changes eventually lead to behavioral abnormalities of individual. Methyl-CpG binding protein 2 (MeCP2), an important transcription inhibitor, contains characteristic do-mains that regulated chromosome conformation, transcription and RNA splicing. It has also been identified that MeCP2 plays an important role in regulating neuronal plasticity and related target gene transcription during brain development, which add more attention about the importance of epigenetic mechanism in neuronal function. Studies have showed that DNA methylation, histone acetylation and phosphorylation regulate MeCP2 gene expression, affect gene and protein transcription, translation and cell regulation in learning, memory and substance addiction. Addictive substances induce psychological and mental dependence, which are related to the changes of neuronal plasticity and gene expression in addictive neural circuits. MeCP2 plays an important role in regulating synaptic transmission and neuronal plasticity in central nervous system. Therefore, it is of great scientific significance to explore the role of MeCP2 in regulating neuronal plasticity in the central nervous system. In this review we summarized the structure and function of MeCP2, the relationship between MeCP2 and epigenetics, and the role of MeCP2 epigenetic modifications induced by different addictive substances in substance addiction, which may provide further understanding of the molecular mechanism of substance addiction and provide new in￣sight for clinical intervention.

Magnetic systems for cancer immunotherapy

Immunotherapy is a rapidly developing area of cancer treatment due to its higher specificity and potential for greater efficacy than traditional therapies. Immune cell modulation through the administration of drugs, proteins, and cells can enhance antitumoral responses through pathways that may be otherwise inhibited in the presence of immunosuppressive tumors. Magnetic systems offer several advantages for improving the performance of immunotherapies, including increased spatiotemporal control over transport, release, and dosing of immunomodulatory drugs within the body, resulting in reduced off-target effects and improved efficacy. Compared to alternative methods for stimulating drug release such as light and pH, magnetic systems enable several distinct methods for programming immune responses. First, we discuss how magnetic hyperthermia can stimulate immune cells and trigger thermoresponsive drug release. Second, we summarize how magnetically targeted delivery of drug carriers can increase the accumulation of drugs in target sites. Third, we review how biomaterials can undergo magnetically driven structural changes to enable remote release of encapsulated drugs. Fourth, we describe the use of magnetic particles for targeted interactions with cellular receptors for promoting antitumor activity. Finally, we discuss translational considerations of these systems, such as toxicity, clinical compatibility, and future opportunities for improving cancer treatment.

Genome-wide association analysis reveals regulation of at-risk loci by DNA methylation in prostate cancer / 亚洲男科学杂志(英文版)

Epigenetic changes are potentially important for the ontogeny and progression of tumors but are not usually studied because of the complexity of analyzing transcript regulation resulting from epigenetic alterations. Prostate cancer (PCa) is characterized by variable clinical manifestations and frequently unpredictable outcomes. We performed an expression quantitative trait loci (eQTL) analysis to identify the genomic regions that regulate gene expression in PCa and identified a relationship between DNA methylation and clinical information. Using multi-level information published in The Cancer Genome Atlas, we performed eQTL-based analyses on DNA methylation and gene expression. To better interpret these data, we correlated loci and clinical indexes to identify the important loci for both PCa development and progression. Our data demonstrated that although only a small proportion of genes are regulated via DNA methylation in PCa, these genes are enriched in important cancer-related groups. In addition, single nucleotide polymorphism analysis identified the locations of CpG sites and genes within at-risk loci, including the 19q13.2-q13.43 and 16q22.2-q23.1 loci. Further, an epigenetic association study of clinical indexes detected risk loci and pyrosequencing for site validation. Although DNA methylation-regulated genes across PCa samples are a small proportion, the associated genes play important roles in PCa carcinogenesis.

Methylated CpG dinucleotides in the 5-α reductase 2 gene may explain finasteride resistance in benign prostatic enlargement patients / 亚洲男科学杂志(英文版)

The inhibition of 5-α reductase type 2 (SRD5A2) by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement (BPE). Certain BPE patients showing no SRD5A2 protein expression are resistant to finasteride therapy. Our previous work showed that methylated cytosine-phosphate-guanine (CpG) islands in the SRD5A2 gene might account for the absence or reduction of SRD5A2 protein expression. Here, we found that the expression of the SRD5A2 protein was variable and that weak expression of the SRD5A2 protein (scored 0-100) occurred in 10.0% (4/40) of benign adult prostates. We showed that the expression of SRD5A2 was negatively correlated with DNA methyltransferase 1 (DNMT1) expression. In vitro SRD5A2-negative BPH-1 cells were resistant to finasteride treatment, and SRD5A2 was re-expressed in BPH-1 cells when SRD5A2 was demethylated by 5-Aza-2'-deoxycytidine (5-Aza-CdR) or N-phthalyl-L-tryptophan (RG108). Furthermore, we determined the exact methylation ratios of CpG dinucleotides in a CpG island of SRD5A2 through MassArray quantitative methylation analysis. Ten methylated CpG dinucleotides, including four CpG dinucleotides in the promoter and six CpG dinucleotides in the first exon, were found in a CpG island located from -400 bp to +600 bp in SRD5A2, which might lead to the silencing of SRD5A2 and the absence or reduction of SRD5A2 protein expression. Finasteride cannot exert a therapeutic effect on patients lacking SRD5A2, which may partially account for the resistance to finasteride observed in certain BPE patients.

Regulation and its mechanism of MeCP2 on biological behavior and epithelial-mesenchymal transition of LECs / 中华实验眼科杂志

Objective@#To investigate the role of methyl-CpG-binding protein 2 (MeCP2) in the regulation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) and its possible mechanism.@*Methods@#Human LEC lines (SRA01/04) were divided into MeCP2-mimic group, MeCP2-NC group and small interferening RNA-MeCP2 (si-MeCP2) group, and MeCP2 analog plasmid, blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection, the migration rate of the cells was evaluated by scratching test, and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions of β-catenin, E-cadherin, Vimentin, matrix metallo proteinase (MMP)-9, MMP-7 and secreted frizzled-related protein 5 (SFRP5) proteins in the cells were detected by Western blot.@*Results@#After 24 hours of transfection, the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group (F=4 773.00, P<0.00 1). The migrating rate of the cells in the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was (57.45±5.20)%, (32.71±10.02)% and (17.77±9.22)%, respectively, showing a significant difference among the three groups (F=124.00, P<0.001), and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group (both at P<0.001). The relative expressing intensity (absorbance) of Wnt3a in the cells of the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was 75.92±6.10, 52.03±5.22 and 28.75±3.39, respectively, with a significant difference among three the groups (F=221.30, P<0.001), and the relative expressing intensity (absorbance) of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group (both at P<0.001). The relative expressing level of E-cadherin protein was significantly elevated and the expressions of β-catenin, Vimentin, MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group (all at P<0.01). The relative expressing level of SFRP5 protein in the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was 27.19±0.03, 47.54±0.05 and 74.93±0.05, respectively, showing a statistical difference among the three groups (F=183.49, P<0.001), and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group (both at P<0.001).@*Conclusions@#MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway.

Regulation and its mechanism of MeCP2 on biological behavior and epithelial-mesenchymal transition of LECs / 中华实验眼科杂志

Objective To investigate the role of methyl-CpG-binding protein 2 (MeCP2) in the regulation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) and its possible mechanism.Methods Human LEC lines (SRA01/04) were divided into MeCP2-mimic group,MeCP2-NC group and small interferening RNA-MeCP2 (si-MeCP2) group,and MeCP2 analog plasmid,blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection,the migration rate of the cells was evaluated by scratching test,and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions of β-catenin,E-cadherin,Vimentin,matrix metallo proteinase (MMP)-9,MMP-7 and secreted frizzled-related protein 5 (SFRP5) proteins in the cells were detected by Western blot.Results After 24 hours of transfection,the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group (F =4 773.00,P＜0.00 1).The migrating rate of the cells in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was (57.45±5.20)％,(32.71± 10.02)％ and (17.77±9.22)％,respectively,showing a significant difference among the three groups (F=124.00,P＜0.001),and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group (both at P＜0.001).The relative expressing intensity (absorbance) of Wnt3a in the cells of the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 75.92 ± 6.10,52.03 ± 5.22 and 28.75 ± 3.39,respectively,with a significant difference among three the groups (F=221.30,P＜0.001),and the relative expressing intensity (absorbance) of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group (both at P＜0.001).The relative expressing level of E-cadherin protein was significantly elevated and the expressions of β-catenin,Vimentin,MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group (all at P＜O.01).The relative expressing level of SFRP5 protein in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 27.19± 0.03,47.54±0.05 and 74.93±0.05,respectively,showing a statistical difference among the three groups (F =183.49,P＜0.001),and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group (both at P＜0.001).Conclusions MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway.

A review of computational algorithms for CpG islands detection

Type II diabetes mellitus (T2DM) and obesity are two common pathophysiological conditions of metabolic syndrome(MetS), a collection of similar metabolic dysfunctions due to sedentary lifestyle and overnutrition. Obesity arises fromimproper adipogenesis which otherwise has a crucial role in maintaining proper metabolic functions. Downstream eventsarising from obesity have been linked to T2DM. The nuclear receptor peroxisome proliferator activator gamma (PPAR-c),responsible for maintaining lipid and glucose homeostasis, is down-regulated under obesity leading to a weakened insulinsensitivity of the human body. In course of our review we will outline details of the down-regulation mechanism, provide anoverview of the current clinical therapeutics and their shortcomings. Toxicity studies on the seminal drug troglitazone,belonging to the most effective glitazone anti-diabetic category, is also discussed. This will lead to an overview aboutstructural adaptations on the existing glitazones to alleviate their side effects and toxicity. Finally, we forward a concept ofnovel therapeutics mimicking the glitazone framework, based on some design concepts and preliminary in silico studies.These could be later developed into dual acting drugs towards alleviating the deleterious effects of obesity on normalglucose metabolism, and address obesity in itself.

Methylation status of the PPP1R13L promoter region among lung cancer patients and healthy controls. Analytical cross-sectional study

ABSTRACT BACKGROUND: There is evidence that genetic predisposition and epigenetic alteration (e.g. DNA methylation) play major roles in lung cancer. In our genetic epidemiological studies, rs1970764 in oncogene PPP1R13L was most consistently associated with lung cancer risk. Here, we explored the role of PPP1R13L methylation in lung cancer development. DESIGN AND SETTING: Analytical cross-sectional study (45 lung cancer cases and 45 controls), conducted in China. METHODS: We investigated the DNA methylation status of 2,160 cytosine-phosphate-guanine (CpG) sites in the PPP1R13L promoter region using the EpiTYPER assay of the Sequenom MassARRAY platform. RESULTS: In the whole study group, the methylation levels of CpG-6, CpG-9, CpG-20 and CpG-21 were significantly lower and those of CpG-16 were significantly higher in cases than in controls. Among smokers, the methylation levels at five CpG sites (CpG-6, CpG-11, CpG-15, CpG-20 and CpG-21) were statistically significantly lower among cases. Among men, the methylation levels at four CpG sites (CpG-11, CpG-15, CpG-20 and CpG-21) were significantly lower among cases. Regarding smokers, the methylation levels at CpG-7.8 and CpG-21 among cases and at CpG-22 among controls were significantly lower, compared with nonsmokers. The frequency of positivity for methylation was not significantly different between lung cancer cases and controls (68.22% for cases and 71.87% for controls; P = 0.119). CONCLUSION: Our study on a Chinese population suggests that lung cancer patients have aberrant methylation status (hypomethylation tended to be more frequent) in peripheral blood leukocytes at several CpG sites in the PPP1R13L promoter region and that exposure to smoking may influence methylation status.

Síndrome de Rett: Análisis molecular del gen MECP2 en pacientes chilenas / Rett syndrome: MECP2 gene molecular analysis in Chilean patients

INTRODUCCIÓN: El síndrome de Rett (RTT) es un trastorno neurológico progresivo caracterizado por producir una regresión del desarrollo psicomotor en niñas previamente sanas. La mayoría de los casos son causados por variantes patogénicas en el gen MECP2, que codifica para la proteína methyl CpG- binding protein 2. OBJETIVO: Describir la frecuencia y el tipo de variantes patogénicas en MECP2 en mujeres chilenas con diagnóstico clínico de RTT. PACIENTES Y MÉTODO: Se invitó a participar en este estudio a mujeres chilenas con sospecha clínica de RTT. Se reunió información clínica mediante un cuestionario. Se analizaron variantes patogénicas en MECP2 mediante el método de secuenciación de Sanger y se utilizó Multiple Ligation-dependant Probe Amplification (MLPA) para la detección de duplicaciones y deleciones. RESULTADO: El estudio incluyó 14 pacientes con sospecha de RTT, de las cuales 8 (57%) pacientes tuvieron variantes patogénicas. Las restantes permanecen sin diagnóstico molecular. CONCLUSIÓN: Variantes patogénicas en MECP2 están presentes en pacientes chilenas con RTT. Es probable que haya otros genes o diagnósticos involucrados en las pacientes sin hallazgos en MECP2. A partir de este trabajo, el diagnóstico molecular está disponible en Chile.

INTRODUCTION: Rett syndrome (RTT) is a progressive neurological disorder characterized by regres sion of psychomotor development in previously healthy girls. Most cases are due to pathogenic va riants in the MECP2 gene which encodes for the methyl CpG-binding protein 2. OBJECTIVE: To des cribe the frequency and type of pathogenic variants in the MECP2 gene in Chilean female patients with clinical diagnosis of RTT. PATIENTS AND METHOD: Chilean women with clinical suspicion of RTT were invited to participate in the study. Clinical data were collected through a questionnaire. MECP2 pathogenic variants were analyzed by Sanger sequencing method and Multiplex Ligation-dependent Probe Amplification (MLPA) was used to detect duplications or deletions. RESULTS: The study in cluded 14 patients with suspected RTT, of which eight (57%) patients had pathogenic variants. The other patients remain without molecular diagnosis. CONCLUSIONS: Pathogenic variants in MECP2 are present in Chilean patients with RTT. It is likely that there are other genes or diagnoses involved in patients without MECP2 findings. As of this study, molecular diagnosis is available in Chile.

AtMBD4: A methylated DNA binding protein negatively regulates a subset of phosphate starvation genes

DNA methylation is an important epigenetic modification that governs transcriptional regulation. The methylation mark isread by a special class of proteins called methyl-CpG-binding domain proteins. The role of DNA methylation has beenfound in X-chromosome inactivation, genomic imprinting, transposon silencing, and self-incompatibility. Recently,remodeling of global DNA methylation was demonstrated in Arabidopsis during low phosphate availability. The presentstudy reports that AtMBD4 gene of Arabidopsis negatively regulates phosphate starvation. The T-DNA insertion mutation atthe AtMBD4 locus exhibited altered root architecture as compared to wild-type plants. Using microarray hybridization andanalysis, an increased transcript accumulation of 242 genes was observed in the mutant. Many of these genes were relatedto phosphate transporters and transcription factors, involved in phosphate starvation response. Comparison of data ofatmbd4 mutant with publicly available microarray data of phosphate starvation response indicated the role of AtMBD4protein in phosphate starvation response. Further, promoter analysis of up-regulated genes suggested that cis-regulatoryelements like MBS, W-box, and B1BS are more prominent in the promoters of up-regulated genes. Upon performing amethylation-specific PCR, a decreased DNA methylation in the promoter regions of up-regulated genes was observed. Theaccumulation of anthocyanin and inorganic phosphate in the atmbd4 mutant was found to be higher than the wild-typeplant. Altered root morphology, up-regulation of phosphate starvation-induced genes in atmbd4 mutant suggests thatAtMBD4 negatively regulates the phosphate starvation response.