Effects of testosterone dose on depression-like behavior among castrated adult male rats.

Previous research has shown a decrease in serum testosterone levels in male patients with depression. In recent years, the results of testosterone replacement therapy (TRT) to improve depression have been mixed. Using the classic CUMS model, we induced depressive-like behaviors in rats and observed a decrease in their serum testosterone levels along with an increase in androgen receptor expression in the hippocampus. We then performed castration and sham surgery on male rats and found that testosterone deprivation led to the manifestation of depressive-like behavior that could be ameliorated by TRT. Through a repeated measures experiment consisting of five blocks over a period of 25 days, we discovered that the reduction in depressive-like behavior in testosterone-deprived rats began 22 days after drug administration (0.5 and 0.25 mg/rat). Furthermore, rats in 0.5mgT group showed the most significant improvements. Subsequently, this dose was used in CUMS rats and reduced the occurrence of depressive-like behaviors. Our study has demonstrated the complex interplay between depression and testosterone, as well as the intricate dose-response relationship between TRT and reduction in depression. Our research supports the use of TRT to alleviate depression, but dosage and duration of treatment are critical factors in determining efficacy.

Single-nucleus transcriptomic analysis reveals the relationship between gene expression in oligodendrocyte lineage and major depressive disorder.

BACKGROUND: Major depressive disorder (MDD) is a common mental illness that affects millions of people worldwide and imposes a heavy burden on individuals, families and society. Previous studies on MDD predominantly focused on neurons and employed bulk homogenates of brain tissues. This paper aims to decipher the relationship between oligodendrocyte lineage (OL) development and MDD at the single-cell resolution level. METHODS: Here, we present the use of a guided regularized random forest (GRRF) algorithm to explore single-nucleus RNA sequencing profiles (GSE144136) of the OL at four developmental stages, which contains dorsolateral prefrontal cortex of 17 healthy controls (HC) and 17 MDD cases, generated by Nagy C et al. We prioritized and ordered differentially expressed genes (DEGs) based on Nagy et al., which could predominantly discriminate cells in the four developmental stages and two adjacent developmental stages of the OL. We further screened top-ranked genes that distinguished between HC and MDD in four developmental stages. Moreover, we estimated the performance of the GRRF model via the area under the curve value. Additionally, we validated the pivotal candidate gene Malat1 in animal models. RESULTS: We found that, among the four developmental stages, the onset development of OL (OPC2) possesses the best predictive power for distinguishing HC and MDD, and long noncoding RNA MALAT1 has top-ranked importance value in candidate genes of four developmental stages. In addition, results of fluorescence in situ hybridization assay showed that Malat1 plays a critical role in the occurrence of depression. CONCLUSIONS: Our work elucidates the mechanism of MDD from the perspective of OL development at the single-cell resolution level and provides novel insight into the occurrence of depression.

Toxicity and Sublethal Effects of Diamide Insecticides on Key Non-Target Natural Predators, the Larvae of Coccinella septempunctata L. (Coleoptera: Coccinellidae).

Coccinella septempunctata (ladybird) is an extremely important natural predator that feeds on aphids. An assessment of the toxicity of pesticides on environmental organisms is an essential component of Integrated Pest Management (IPM) strategies. This study evaluated diamide insecticides' toxicity at lethal and 30% lethal doses (LR30) against C. septempunctata larvae. The pre-imaginal median lethal doses (LR50) of chlorantraniliprole 10% SC, tetrachlorantraniliprole 10% SC, and broflanilide 10% SC were calculated to be 42.078, 289.516, and 0.0943 g active ingredient (a.i.)/ha, respectively. The mortality tests demonstrated that chlorantraniliprole and tetrachlorantraniliprole are comparatively less toxic to C. septempunctata than broflanilide, which were detected to be highly toxic to C. septempunctata. The mortality rates of the groups treated with the three diamide insecticides tended to stabilize after 96 h, extending to the pre-imaginal stage. Furthermore, when compared to broflanilide, which had a much higher potential risk, the hazard quotient (HQ) values indicated that chlorantraniliprole and tetrachlorantraniliprole have a lower risk potential for C. septempunctata in farmland and off farmland. The LR30 dose induces abnormalities in the development phase 4th-instar larvae weight, pupal weight, and adult weight of treated C. septempunctata. The study emphasizes the importance of assessing the adverse effects of diamide insecticides on natural predator species that serve as biological control agents in agricultural IPM strategies.

Tunicamycin induces depression-like behaviors in male rats, accompanied by initiated chaperon-mediated autophagy and decreased synaptic protein expression in the hippocampus.

BACKGROUND AND AIM: Endoplasmic reticulum (ER) stress participates in the occurrence and development of depression, but the underlying mechanism is not fully understood. This study aimed to investigate the behavioral performance and intracerebral molecular changes in an ER stress model of male rats. METHODS: Intrahippocampal injection of tunicamycin (TM) was performed on male rats as a model of ER stress. The body weight was determined, and behavioral tests, including sucrose preference test (SPT), open field test (OFT), and forced swimming test (FST), were performed to evaluate depressive and anxiety-like phenotypes within 8 days after injection. The levels of chaperone-mediated autophagy (CMA), synaptic proteins, and neuroinflammation related factors in this model were measured via real-time quantitative PCR and Western blot analysis. RESULTS: Intrahippocampal injection of TM (2 or 1 µg) induced depression-like behaviors in rats, as indicated by the reduced body weight, sucrose preference in SPT, central time in OFT, and increased immobility time in FST. The mRNA and protein levels of GRP78, ATF4, CHOP, LAMP2A, IL-1ß, IL-6, and TNF-α were significantly increased, while the expressions of MEF2D, PSD95, SYN, p-CREB (Ser133), and BDNF were significantly decreased in the hippocampus in the model group compared with the sham group. CONCLUSIONS: These results confirmed that intrahippocampal injection of TM was a valid method to induce an ER stress rat model with depression-like behaviors accompanied by decreased synaptic protein expression and neuroinflammation. The alteration in CMA-related proteins in this ER stress depression model indicated the involvement of CMA in the development of depression.

Social isolation after chronic unpredictable mild stress perpetuates depressive-like behaviors, memory deficits and social withdrawal via inhibiting ERK/KEAP1/NRF2 signaling.

BACKGROUND: Social withdrawal in patients with depression can aggravate depressive symptoms. However, few studies focus on the behavioral changes of social isolation after CUMS. NRF2 had been reported to be down-regulated after CUMS. But whether NRF2 participates in behavioral changes induced by social isolation after CUMS remains unclear. This study aims to develop a new model combined social isolation with CUMS, and investigate whether such behavioral changes are related to NRF2 signaling. METHODS: This study included two stages. In Stage 1, rats were subjected to 4-week CUMS and CUMS-susceptible rats were selected. In Stage 2, the CUMS-susceptible rats received 4-week social isolation or social support. Behavioral tests were carried out to observe behavioral changes, including sucrose preference test, forced swimming test, open field test, novel object recognition and social interaction test. QRT-PCR, western blot and immunofluorescence staining detected the ERK/KEAP1/NRF2 signaling. RESULTS: CUMS-susceptible rats exhibited depressive-like behaviors accompanied by the down-regulated ERK/KEAP1/NRF2 signaling in hippocampus. In Stage 2, compared with 4-week social support (group CUMSG), 4-week social isolation (group CUMSI) perpetuated the depressive-like behaviors, memory deficits and social withdrawal in CUMS-susceptible rats, as well as lower levels of p-ERK, NRF2, p-NRF2, HO-1 and NQO1, and the higher levels of KEAP1 in hippocampus. CONCLUSION: These findings suggested that social isolation after CUMS perpetuated depressive-like behaviors, memory deficits and social withdrawal via inhibiting ERK/KEAP1/NRF2 signaling. This study provided molecular evidence for the effects of post-stress social isolation on mental health, and the antioxidant stress signaling might be a target to rescue these.

Polydatin Prevents Neuroinflammation and Relieves Depression via Regulating Sirt1/HMGB1/NF-κB Signaling in Mice.

Depression is a prevalent psychiatric disorder with a significant health impact and economic burden worldwide. Unfortunately, the exact pathogenesis of depression is not well understood. Neuroinflammation and microglial activation play an essential role in the pathogenesis of depression. Previous studies have shown that polydatin has anti-inflammatory and antioxidant properties. However, the link between polydatin and depression remains unclear. Therefore, the objective of this study was to investigate the antidepressant effect of polydatin in lipopolysaccharide (LPS)-induced depression in mice and its possible mechanism. Adult male C57BL/6 J mice were used in this study. The polydatin and LPS were injected intraperitoneally daily for 5 days. In addition, the EX527, an inhibitor of Sirt1, was injected intraperitoneally daily and 1 h before the polydatin injection. The behavior tests were performed to elucidate the depression-like behaviors. The Sirt1/HMGB1/NF-κB pathway expression was detected by western blot, ELISA, and immunofluorescence staining. Polydatin can significantly improve LPS-induced depression-like behavior in mice. Treatment with polydatin increased the expression of the Sirt1 but decreased the expression of the HMGB1, p-NF-κB, IL-1b, and TNF-α in the LPS-induced depression mice. In addition, the EX527 abolished the anti-depressive effects of the polydatin and the levels of Sirt1 protein. These findings suggested that the polydatin reversed the depressive effects through the Sirt1/HMGB1/NF-κB signaling in the LPS-induced depression mice. Therefore, polydatin can be used in the treatment of depression.

CLDN6 inhibits breast cancer cell malignant behavior by suppressing ERK signaling.

Claudin 6 (CLDN6) is an important component of tight junctions. Through the PDZ binding motif, CLDN6 binds to a variety of signaling proteins that contain the PDZ domain to regulate different signaling pathways, and plays an important role in the occurrence and development of tumors. Our previous work showed that CLDN6 was expressed at low levels in breast cancer cells, and overexpression of CLDN6 inhibited breast cancer cell proliferation, migration and invasion. However, the mechanism of how CLDN6 works remains unclear. In this study, we aimed to explore the mechanism by which CLDN6 inhibits breast cancer cell malignant behavior. As a result, overexpression of CLDN6 inhibited the proliferation of breast cancer cells along with the downregulation of cyclin D1, which plays an important role in regulating cell proliferation. After overexpression of Sp1 in CLDN6-overexpressing cells, the expression of cyclin D1 was upregulated. On the other hand, CLDN6 inhibited breast cancer cell migration and invasion along with the downregulation of IL-8, CXCR2 and FAK. When treated with IL-8, the migration and invasion ability were promoted along with the upregulation of CXCR2 and p-FAK, and the cytoskeleton was rearranged in CLDN6-overexpressing cells. Furthermore, when treated with the ERK signaling activator PMA, the proliferation, migration and invasion abilities were promoted along with the upregulation of Sp1, cyclin D1 and IL-8 in CLDN6-overexpressin cells. In conclusion, CLDN6 suppressed ERK/Sp1/cyclin D1 and ERK/IL-8 signaling to inhibit proliferation, migration and invasion in breast cancer cells. The mechanism may provide experimental evidence for the treatment of breast cancer targeting CLDN6.

RGFP966 is protective against lipopolysaccharide-induced depressive-like behaviors in mice by inhibiting neuroinflammation and microglial activation.

Depression is a prevalent mental disorder. However, its pathophysiological mechanism has still remained elusive, and a limited number of effective treatments have been presented. Recent studies have shown that neuroinflammation and microglial activation are involved in the pathogenesis of depression. Histone deacetylase 3 (HDAC3) has neurotoxic effects on several neuropathological conditions. The inhibition of HDAC3 has been reported to induce anti-inflammatory and antioxidant effects. RGFP966 is a highly selective inhibitor of HDAC3. This study aimed to investigate the antidepressant effect of RGFP966 on lipopolysaccharide (LPS)-induced depressive-like behaviors in mice and to explore its possible mechanism. Adult male C57BL/6J mice were utilized in this study. The LPS and RGFP966 were injected intraperitoneally daily for 5 days. The behavior tests were performed to elucidate the depression-like behaviors. Western blot, ELISA and immunofluorescence staining were used to study the HDAC3/TLR4/NLRP3 pathway-related proteins. The results of behavioral tests showed that RGFP966 could improve the LPS-induced depressive-like behaviors in mice. The results of Western blotting showed that RGFP966 treatment downregulated the expression levels of toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3), caspase-1, and interleukin-1ß (IL-1ß) (P < 0.05). Furthermore, the results of immunofluorescence staining showed that RGFP966 treatment inhibited microglial activation in the hippocampus of mice (P < 0.01). These findings suggested that RGFP966 could effectively ameliorate LPS-induced depressive-like behaviors in mice by inhibiting neuroinflammation and microglial activation. The anti-inflammatory mechanism of RGFP966 might be related to the inhibition of the HDAC3/TLR4/NLRP3 signaling pathway. Therefore, inhibition of HDAC3 using RGFP966 could serve as a potential treatment strategy for depression.

Possible Vicarious Traumatization Among Psychiatric Inpatients During the Remission Phase of the COVID-19: A Single-Center Cross-Sectional Study.

Background: Far from being a clinical disease, the COVID-19 pandemic has become a threatening social event worldwide exerting long-term impacts on human beings. Objective: This study was designed to determine if and to what extent psychiatric inpatients during the remission phase of the pandemic suffered from vicarious traumatization. Method: Totally 266 eligible participants from psychiatric and psychological wards in a hospital were recruited during October 26th, 2020 to February 4th, 2021 to finish a self-made online questionnaire consisting of Impact of Event Scale-Revised (IES-R), Self-Rating Depression Scale (SDS), Self-Rating Anxiety Scale (SAS), Obsessive-Compulsive Inventory-Revised (OCI-R), Pittsburgh Sleep Quality Index-Revised (PSQI-R), Social Support Rating Scale (SSRS), Beck Suicide Ideation Scale (SSI), 12-Item Short-Form Health Survey (SF-12). Meanwhile, some socio-demographics and information related to the pandemic were also recorded. Results: The detection rate of vicarious traumatic symptoms (VTS) was 80.83%, including 40.98% for mild ones, 25.56% for moderate ones, and 14.29% for severe ones, among whom 98.14% possessed all three phenotypes. 27.07% of the sample were considered possible vicarious traumatization (pVT). Having acquaintances infected with or died from COVID-19, worries on re-outbreak of COVID-19, a higher score of OCI-R or lower score of SF-12, and long latency of VTS were independent risk factors of pVT. Conclusion: Our study showed that COVID-19 could have profound mental influences on psychiatric inpatients. It is high time we did some screening in the wards to seek for patients at risk.

Oridonin ameliorates insulin resistance partially through inhibition of inflammatory response in rats subjected to chronic unpredictable mild stress.

BACKGROUND: Oridonin (Ori) has multiple biological properties, especially anti-inflammatory. However, its effects on chronic unpredictable mild stress (CUMS)-induced insulin resistance are still unclear. In this study, we explored the regulatory role of Ori in CUMS-triggered insulin resistance, and the underlying molecular mechanisms; Methods: SD rats were subjected to CUMS for 4 weeks, some of which were injected with Ori or fluoxetine (FLX) in durations of CUMS. After CUMS procedure, the behavioral and metabolic tests were performed. Elisa, immunofluorescence and western blotting were used to determine the inflammatory response and NLRP3 inflammasome activation. We investigated the interaction between NLRP3 and NEK7 using immunoprecipitation. Finally, we detected the proinflammatory cytokines in Lipopolysaccharide (LPS)-activated RAW264.7 cells treated with Ori; RESULTS: In this study, we found that chronic stress resulted in depressive-like behavior comorbid with insulin resistance. Ori was discovered to ameliorate insulin resistance as well as insulin signaling disturbance in the hippocampus. In addition, CUMS caused the infiltration of macrophages into the islets. And IL-1ß, IL-18 and caspase-1 were elevated in pancreases of CUMS rats, which could also be reversed by Ori treatment via reducing the interaction between NLRP3 and NEK7. Furthermore, Ori dose-dependently inhibited the levels of IL-1ß and IL-18 in LPS-activated RAW264.7 cells; CONCLUSIONS: All these results supported our hypothesis that Ori possesses potent anti-insulin resistant actions, which is partially correlated with inhibiting infiltration of macrophages into the islets and NLRP3 activation induced by CUMS. Therefore, our results highlighted the protective role of Ori against CUMS-elicited insulin resistance.

Integrated Analysis of Methylomic and Transcriptomic Data to Identify Potential Diagnostic Biomarkers for Major Depressive Disorder.

Major depressive disorder (MDD) is a mental illness with high incidence and complex etiology, that poses a serious threat to human health and increases the socioeconomic burden. Currently, high-accuracy biomarkers for MDD diagnosis are urgently needed. This paper aims to identify novel blood-based diagnostic biomarkers for MDD. Whole blood DNA methylation data and gene expression data from the Gene Expression Omnibus database are downloaded. Then, differentially expressed/methylated genes (DEGs/DMGs) are identified. In addition, we made a systematic analysis of the DNA methylation on 5'-C-phosphate-G-3' (CpGs) in all of the gene regions, as well as different gene regions, and then we defined a "dominant" region. Subsequently, integrated analysis is employed to identify the robust MDD-related blood biomarkers. Finally, a gene expression classifier and a methylation classifier are constructed using the random forest algorithm and the leave-one-out cross-validation method. Our results demonstrate that DEGs are mainly involved in the inflammatory response-associated pathways, while DMGs are primarily concentrated in the neurodevelopment- and neuroplasticity-associated pathways. Our integrated analysis identified 46 hypo-methylated and up-regulated (hypo-up) genes and 71 hyper-methylated and down-regulated (hyper-down) genes. One gene expression classifier and two DNA methylation classifiers, based on the CpGs in all of the regions or in the dominant regions are constructed. The gene expression classifier possessed the best predictive ability, followed by the DNA methylation classifiers, based on the CpGs in both the dominant regions and all of the regions. In summary, the integrated analysis of DNA methylation and gene expression has identified 46 hypo-up genes and 71 hyper-down genes, which could be used as diagnostic biomarkers for MDD.

Prevalence and Characteristics of Obsessive-Compulsive Disorder Among Urban Residents in Wuhan During the Stage of Regular Control of Coronavirus Disease-19 Epidemic.

Background: Coronavirus disease-19 (Covid-19) is one of the most devastating epidemics in the 21st century, which has caused considerable damage to the physical and mental health of human beings. Despite a few regions like China having controlled the epidemic trends, most countries are still under siege of COVID-19. As the emphasis on cleaning and hygiene has been increasing, the problems related to obsessive-compulsive disorder (OCD) may appear. Objective: This study was designed to investigate the prevalence of OCD in the urban population in Wuhan during the stage of regular epidemic control and prevention. Meanwhile, characteristics and risk factors for OCD were also explored. Method: Five-hundred and seventy residents in urban areas of Wuhan were recruited using the snowball sampling method to complete questionnaires and an online interview from July 9 to July 19, 2020. Collected information encompassed socio-demographics, Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) scores, Social Support Rating Scale (SSRS) scores and Pittsburgh Sleep Quality Index(PSQI) values. Results: Three months after lifting the quarantine in Wuhan, the prevalence of OCD was 17.93%. About 89% of OCD patients had both obsessions and compulsions, while 8% had only obsessions and 3% had only compulsions. Top 3 common dimensions of obsessions were miscellaneous (84.0%), aggressive (76.6%), and contamination (48.9%), and of compulsions were miscellaneous (64%), checking (51.7%), and cleaning/washing/repeating (31.5%). The unmarried were more vulnerable to OCD than the married (p < 0.05, odds ration = 1.836). Students had 2.103 times the risk of developing OCD than health care workers (p < 0.05). Those with positive family history of OCD and other mental disorders (p < 0.05, odds ration = 2.497) and presence of psychiatric comorbidity (p < 0.05, odds ration = 4.213) were also at higher risk. Each level increase in sleep latency increased the risk of OCD to 1.646 times (p < 0.05). Conclusion: In the background of regular epidemic control, the prevalence of OCD was high, and the symptoms were widely distributed. Obsessions often accompanied compulsions. Being single and a student, positive family history of OCD and other mental disorders, presence of psychiatric comorbidity, and longer sleep latency were predictors of OCD. Early recognition and detection of these issues may help to intervene in OCD.

EZH2 RIP-seq Identifies Tissue-specific Long Non-coding RNAs.

BACKGROUND: Polycomb Repressive Complex 2 (PRC2) catalyzes histone methylation at H3 Lys27, and plays crucial roles during development and diseases in numerous systems. Its catalytic subunit EZH2 represents a key nuclear target for long non-coding RNAs (lncRNAs) that emerging to be a novel class of epigenetic regulator and participate in diverse cellular processes. LncRNAs are characterized by high tissue-specificity; however, little is known about the tissue profile of the EZH2- interacting lncRNAs. OBJECTIVE: Here we performed a global screening for EZH2-binding lncRNAs in tissues including brain, lung, heart, liver, kidney, intestine, spleen, testis, muscle and blood by combining RNA immuno- precipitation and RNA sequencing. We identified 1328 EZH2-binding lncRNAs, among which 470 were shared in at least two tissues while 858 were only detected in single tissue. An RNA motif with specific secondary structure was identified in a number of lncRNAs, albeit not in all EZH2-binding lncRNAs. The EZH2-binding lncRNAs fell into four categories including intergenic lncRNA, antisense lncRNA, intron-related lncRNA and promoter-related lncRNA, suggesting diverse regulations of both cis and trans-mechanisms. A promoter-related lncRNA Hnf1aos1 bound to EZH2 specifically in the liver, a feature same as its paired coding gene Hnf1a, further confirming the validity of our study. In addition to the well known EZH2-binding lncRNAs like Kcnq1ot1, Gas5, Meg3, Hotair and Malat1, majority of the lncRNAs were firstly reported to be associated with EZH2. CONCLUSION: Our findings provide a profiling view of the EZH2-interacting lncRNAs across different tissues, and suggest critical roles of lncRNAs during cell differentiation and maturation.

CLDN6 enhances chemoresistance to ADM via AF-6/ERKs pathway in TNBC cell line MDAMB231.

Claudin-6 (CLDN6), a critical tight junction protein acting as a tumor suppressor in breast cancer, is also considered to be a stem cell marker. Triple-negative breast cancer (TNBC) is a subtype of claudin-low and stem cell-like breast cancer which is chemoresistant to multiple anti-cancer drugs. The aim of our study was to determine whether CLDN6 plays a role in chemoresistance of TNBC. We found that overexpression of CLDN6 in TNBC cell line MDAMB231 significantly inhibited cell growth, migration, and invasion. The expression of CLDN6 increased the IC50 of adriamycin (ADM) and promoted the clonogenic survival. CLDN6 inhibited ADM-induced apoptosis and senescence in MDAMB231 cells. However, P-gp, a resistance-related protein highly associated with chemoresistance, was downregulated by CLDN6 overexpression in MDAMB231 cells. Epithelial mesenchymal transition (EMT) marker E-cadherin was increased, and vimentin was decreased by CLDN6. In addition, stem cell markers OCT4, SOX2, and Nanog were dramatically increased. CLDN6 colocalized and interacted with AF-6. Overexpression of CLDN6 increased the expression of afadin (AF-6) and hampered the activation of ERK signaling. PMA, a specific ERK activator, reversed the expression of EMT and stem cell markers, and decreased chemoresistance of MDAMB231 cells to ADM with a decreased IC50 and an increased apoptosis resulting from CLDN6. Together, we conclude that CLDN6 enhances the chemoresistance to ADM via activating the AF-6/ERK signaling pathway and up-regulating cancer stem cell characters in MDAMB231 cells.

Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions.

Claudin-6 (CLDN6) is an integral component of the tight junction proteins in polarized epithelial and endothelial cells and plays a crucial role in maintaining cell integrity. Deregulation of CLDN6 expression and distribution in tumor tissues have been widely documented and correlated with cancer progression and metastasis. However, a complete mechanistic understanding of CLDN6 regulation and function remains to be studied. Herein, we show new potential properties of CLDN6 regulation and functions from bioinformatics analysis. Using numerous algorithms to characterize the CLDN6 gene promoter elements and the CLDN6 protein structure, physio-chemical and localization properties, and its evolutionary relationships. CLDN6 is regulated by a diverse set of transcription factors (SP1, SPR, AML-1a, CdxA, CRE-BP and CREB) and associated with the levels of methylation of CpG islands in promoters. The structural properties of CLDN6 indicate that it promotes cancer cell behavior via the ASK1-p38/JNK MAPK secretory signaling pathway. In conclusion, this information from bioinformatics analysis will help future attempts to better understand CLDN6 regulation and functions.

DNA methylation of claudin-6 promotes breast cancer cell migration and invasion by recruiting MeCP2 and deacetylating H3Ac and H4Ac.

BACKGROUND: Claudin-6 (CLDN6), a member of claudin transmembrane protein family, has recently been reported to be undetectable or at low levels in human breast cancer cell lines and tissues and plays a role in suppression of migration and invasion in breast cancer cells. In addition, it is reported that CLDN6 expression is regulated by DNA methylation in various human cancers and cell lines. However, it is unclear how DNA methylation regulates CLDN6 expression. Here we show the mechanism by which DNA methylation regulates CLDN6 expression in human breast cancer cell line MCF-7. METHODS: RT-PCR, Western blot and immunofluorescent staining were utilized to investigate CLDN6 expression in breast cancer tissues and MCF-7 cells. Methylation-Specific PCR (MSP) was applied to determine DNA methylation status in CLDN6 gene promoter region. Wound-healing assay and invasion assay were utilized to test mobility of MCF-7 cells treated with 5-aza-dC (DNA methyltransferase inhibitor). MeCP2 binding, H3Ac and H4Ac in CLDN6 promoter region were analyzed by ChIP assay. Nuclease accessibility assay was performed for analysis of the chromatin conformation of CLDN6 gene. To study the role of CLDN6 in malignant progression, we used RNAi to knockdown CLDN6 expression in MCF-7 cells treated with 5-aza-dC, and examined the mobility of MCF-7 cells by wound-healing assay and invasion assay. RESULTS: 5-aza-dC and TSA (histone deacetylase inhibitor) application induced CLDN6 expression in MCF-7 cells respectively and synergistically. 5-aza-dC treatment induced CLDN6 demethylation, inhibited MeCP2 binding to CLDN6 promoter and increased H3Ac and H4Ac in the promoter. In addition, TSA increased H4Ac, not H3Ac in the promoter. The chromatin structure of CLDN6 gene became looser than the control group after treating with 5-aza-dC in MCF-7 cells. 5-aza-dC up-regulated CLDN6 expression and suppressed migration and invasion in MCF-7 cells, whereas CLDN6 silence restored tumor malignance in MCF-7 cells. CONCLUSIONS: DNA methylation down-regulates CLDN6 expression through MeCP2 binding to the CLDN6 promoter, deacetylating H3 and H4, and altering chromatin structure, consequently promoting migratory and invasive phenotype in MCF-7 cells.

Compressed matrix thin film (CMTF)-assisted laser desorption ionization mass spectrometric analysis.

The inhomogeneous re-crystallization process of matrix materials is the major concerns associated with matrix assisted laser desorption/ionization (MALDI) analysis. We describe here the approach termed compressed matrix thin film (CMTF) in order to make a uniform matrix deposition. In this approach, solid matrix particles are compressed under 10 MPa of pressure by a compressor that is regularly used in infrared spectroscopic analysis. Then aqueous samples can be deposited on the surface of the matrix film. Major advantages of the CMTF approach are summarized as follows. (1) Reproducible sample preparation procedure. Size and thickness of matrix thin films can be controlled by using a fixed mold.force and known amount of matrix materials. (2) Significantly decreased shot-to-shot variations and enhanced reproducibility. (3) Tolerance for in situ salt washing. Because matrix materials are hydrophobic, salts can be washed away while proteins or peptides are retained on the surface of matrix thin films through hydrophobic interactions. (4) Improved sensitivity. The hydrophobic coating of MALDI sample plate by matrix thin films prevents the spreading of samples across the plate and confines analytes to a small area, leading to increased local concentration. (5) A new means for tissue analysis. Tissue sections can be directly transferred to the uniform surface of matrix materials for reproducible and quantitative comparison of different molecules in different localization. The proposed CMTF should be an enabling technique for mass spectrometric analysis with improved correlations between signal intensities and sample quantities.