Abundance of Modifications in Mature miRNAs Revealed by LC-MS/MS Method Coupled with a Two-Step Hybridization Purification Strategy.

Accurate detection of endogenous miRNA modifications, such as N6-methyladenosine (m6A), 7-methylguanosine (m7G), and 5-methylcytidine (m5C), poses significant challenges, resulting in considerable uncertainty regarding their presence in mature miRNAs. In this study, we demonstrate for the first time that liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) nucleoside analysis method is a practical tool for quantitatively analyzing human miRNA modifications. The newly designed liquid-solid two-step hybridization (LSTH) strategy enhances specificity for miRNA purification, while LC-MS/MS offers robust capability in recognizing modifications and sufficient sensitivity with detection limits ranging from attomoles to low femtomoles. Therefore, it provides a more reliable approach compared to existing techniques for revealing modifications in endogenous miRNAs. With this approach, we characterized m6A, m7G, and m5C modifications in miR-21-5p, Let-7a/e-5p, and miR-10a-5p isolated from cultured cells and observed unexpectedly low abundance (<1% at each site) of these modifications.

FBXW7 loss of function promotes esophageal squamous cell carcinoma progression via elevating MAP4 and ERK phosphorylation.

BACKGROUND: Increasing evidence suggests that FBXW7 has a high frequency of mutations in esophageal squamous cell carcinoma (ESCC). However, the function of FBXW7, especially the mutations, is not clear. This study was designed to investigate the functional significance of FBXW7 loss of function and underlying mechanism in ESCC. METHODS: Immunofluorescence was applied to clarify the localization and main isoform of FBXW7 in ESCC cells. Sanger sequencing were performed to explore mutations of FBXW7 in ESCC tissues. Proliferation, colony, invasion and migration assays were performed to examine the functional roles of FBXW7 in ESCC cells in vitro and in vivo. Real-time RT-PCR, immunoblotting, GST-pulldown, LC-MS/MS and co-immunoprecipitation assay were used to explore the molecular mechanism underlying the actions of FBXW7 functional inactivation in ESCC cells. Immunohistochemical staining were used to explore the expression of FBXW7 and MAP4 in ESCC tissues. RESULTS: The main FBXW7 isoform in ESCC cells was the ß transcript in the cytoplasm. Functional inactivation of FBXW7 led to activation of the MAPK signaling pathway and upregulation of the downstream MMP3 and VEGFA, which enhanced tumor proliferation cell invasion and migration. Among the five mutation forms screened, S327X (X means truncated mutation) had an effect similar to the FBXW7 deficiency and led to the inactivation of FBXW7 in ESCC cells. Three other point mutations, S382F, D400N and R425C, attenuated but did not eliminate FBXW7 function. The other truncating mutation, S598X, which was located outside of the WD40 domain, revealed a tiny attenuation of FBXW7 in ESCC cells. Notably, MAP4 was identified as a potential target of FBXW7. The threonine T521 of MAP4, which was phosphorylated by CHEK1, played a key role in the FBXW7-related degradation system. Immunohistochemical staining indicated that FBXW7 loss of function was associated with tumor stage and shorter survival of patients with ESCC. Univariate and multivariate Cox proportional hazards regression analyses showed that high FBXW7 and low MAP4 was an independent prognostic indicator and prospective longer survival. Moreover, a combination regimen that included MK-8353 to inhibit the phosphorylation of ERK and bevacizumab to inhibit VEGFA produced potent inhibitory effects on the growth of FBXW7 inactivation xenograft tumors in vivo. CONCLUSIONS: This study provided evidence that FBXW7 loss of function promoted ESCC via MAP4 overexpression and ERK phosphorylation, and this novel FBXW7/MAP4/ERK axis may be an efficient target for ESCC treatment.

A Multi-Scale Study on Deformation and Failure Process of Metallic Structures in Extreme Environment.

It is a macro-micro model study for defect initiation, growth and crack propagation of metallic truss structure under high engine temperature and pressure conditions during the reentry atmosphere. Till now, the multi-scale simulation methods for these processes are still unclear. We explore the deformation and failure processes from macroscale to nanoscale using the Gas-Kinetic Unified Algorithm (GKUA) and all-atomic, molecular dynamic (MD) simulation method. The behaviors of the dislocations, defect evolution and crack propagation until failure for Aluminum-Magnesium (Al-Mg) alloy are considered with the different temperature background and strain fields. The results of distributions of temperature and strain field in the aerodynamic environment obtained by molecular dynamics simulations are in good agreement with those obtained from the macroscopic Boltzmann method. Compared to the tensile loading, the alloy structure is more sensitive to compression loading. The polycrystalline Al-Mg alloy has higher yield strength with a larger grain size. It is due to the translation of plastic deformation mode from grain boundary (GB) sliding to dislocation slip and the accumulation of dislocation line. Our findings have paved a new way to analyze and predict the metallic structural failure by micro-scale analysis under the aerodynamic thermal extreme environment of the reentry spacecraft on service expiration.

SUMOylation of Translationally Regulated Tumor Protein Modulates Its Immune Function.

Translationally controlled tumor protein (TCTP) is a highly conserved protein possessing numerous biological functions and molecular interactions, ranging from cell growth to immune responses. However, the molecular mechanism by which TCTP regulates immune function is largely unknown. Here, we found that knockdown of Bombyx mori translationally controlled tumor protein (BmTCTP) led to the increased susceptibility of silkworm cells to virus infection, whereas overexpression of BmTCTP significantly decreased the virus replication. We further demonstrated that BmTCTP could be modified by SUMOylation molecular BmSMT3 at the lysine 164 via the conjugating enzyme BmUBC9, and the stable SUMOylation of BmTCTP by expressing BmTCTP-BmSMT3 fusion protein exhibited strong antiviral activity, which confirmed that the SUMOylation of BmTCTP would contribute to its immune responses. Further work indicated that BmTCTP is able to physically interact with interleukin enhancer binding factor (ILF), one immune molecular, involved in antivirus, and also induce the expression of BmILF in response to virus infection, which in turn enhanced antiviral activity of BmTCTP. Altogether, our present study has provided a novel insight into defending against virus via BmTCTP SUMOylation signaling pathway and interacting with key immune molecular in silkworm.

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10-5 s-1), which interprets its quite low binding energy (-54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

Development of a novel, fully human, anti-PCSK9 antibody with potent hypolipidemic activity by utilizing phage display-based strategy.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favourable druggability by utilizing phage display-based strategy. METHODS: A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human scFv phage display library with hPCSK9, and performing two in vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a full-length Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity. FINDINGS: Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a KD as low as 1.42 nM, and a dramatically slow dissociation rate (koff, 4.68 × 10-6 s-1), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P = 0.658, unpaired Student's t-test), 30.2% (P = 0.002, Mann-Whitney U-test) and 37.2% (P = 0.002, Mann-Whitney U-test), respectively. INTERPRETATION: FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.

MicroRNA-155-5p/EPAS1/interleukin 6 pathway participated in the protection function of sphingosylphosphorylcholine to ischemic cardiomyocytes.

AIM: Previous research in our laboratory found that a biologically active sphingomyelin metabolite, sphingosylphosphorylcholine (SPC), can inhibit myocardial cell apoptosis caused by ischemia with an unknown mechanism. Here, we aimed to study the possible participation of EPAS1 in the protection process of SPC. METHODS: The rat cardiomyocytes deprived of serum were used to mimic ischemic-caused apoptosis, then treated with or without SPC. The expression and nuclear shift of EPAS1 were detected by western blot and immunofluorescence, and its function was studied using its siRNA. KEY FINDING: Our research shows that SPC inhibited serum starvation caused cardiomyocyte apoptosis, accompanied by the up-regulation and nucleus translocation of EPAS1. EPAS1 levels did not change when its transcript was blocked by Actinomycin D, which prompted us to search for a post-transcription mechanism for its increased expression, and finally found that miR-155-5p, regulated by STAT3, was a new post-transcription regulator to EPAS1. Further investigation found that EPAS1 participated in the protective effect of SPC is mainly achieved by activating the downstream target gene, interleukin-6 (IL-6). SIGNIFICANCE: Our results expand our understanding of the biological functions of SPC, and bring a new pathway as a potential therapeutic target to the treatment of cardiovascular diseases caused by myocardial apoptosis.

Sphingosylphosphorylcholine alleviates hypoxia-caused apoptosis in cardiac myofibroblasts via CaM/p38/STAT3 pathway.

Blockade of hypoxia-caused nonmyocytes apoptosis helps improve survival and mitigate ventricular remodeling and dysfunction during the chronic stage of myocardial infarction. But tools affecting nonmyocyte apoptosis are very rare. Sphingosylphosphorylcholine (SPC), a naturally occurring bioactive sphingolipid in plasma, was proved to protect cardiomyocyte against apoptosis in an ischemic model in our previous study. Here, we showed that SPC also inhibited hypoxia-induced apoptosis in myofibroblasts, an important type of nonmyocytes in the heart. Calmodulin (CaM) is an identified receptor of SPC. We clarified that SPC inhibited myofibroblast apoptosis through CaM as evidenced by decreased cleaved caspase 3, PARP1 and condensed nucleus. Furthermore, the employment of inhibitor and agonist of p38 and STAT3 suggests that SPC inhibits myofibroblast apoptosis by regulating the phosphorylation of p38 and STAT3, and they act as downstream of CaM. The present work may provide new evidence on the regulation of myofibroblasts apoptosis by SPC and a novel target for heart remodeling after hypoxia.

Gut microbiota dysbiosis-induced activation of the intrarenal renin-angiotensin system is involved in kidney injuries in rat diabetic nephropathy.

Some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (DM). In this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (DN) and its underlying mechanisms. Male SD rats were intraperitoneally injected with streptozotocin to induce DM. DM rats were orally administered compound broad-spectrum antibiotics for 8 weeks. After the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. We found that compared with the control rats, DM rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. Furthermore, the protein levels of angiotensin II, angiotensin-converting enzyme, and angiotensin II type 1 receptor in the kidneys of DM rats were significantly increased. Administration of broad-spectrum antibiotics in DM rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal RAS activation, and attenuated kidney damage. Finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin II protein expression (r = 0.969, P < 0.001). In conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early DN through activating intrarenal RAS.

Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System.

: Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

Urinary levels of podocyte-derived microparticles are associated with the progression of chronic kidney disease.

BACKGROUND: Podocyte-derived microparticles (MPs) could be secreted from activated or apoptotic podocytes. An increased number of podocyte-derived MPs in the urine might reflect podocyte injury in renal diseases. This study aimed to observe the change of urinary podocyte-derived MP levels in patients with chronic kidney disease (CKD) and to further explore its correlation with the progression of CKD. METHODS: A prospective, longitudinal study was conducted in eighty patients with biopsy-proven CKD. Podocyte-derived MPs (annexin V and podocalyxin positive) were detected by flow cytometry. The number of urinary podocyte-derived MPs was analyzed to evaluate the association with biochemical measurements and pathological glomerulosclerosis assessment. Patients with idiopathic membranous nephropathy (IMN) were followed up after the six-month treatment of prednisone combined with tacrolimus to evaluate the association of urinary podocyte-derived MP levels and the remission of IMN. RESULTS: The CKD patients had higher urinary podocyte-derived MP levels compared with healthy controls (HCs). Baseline urinary levels of podocyte-derived MPs were positively correlated with 24-hour proteinuria, while were inversely correlated with the percentage of global glomerulosclerosis. The urinary podocyte-derived MPs levels had good discrimination for glomerulosclerosis [area under curve (AUC), 0.66]. The urinary podocyte-derived MPs levels in IMN patients were significantly decreased accompanied with the recovery of abnormal clinical parameters after six-month treatment. CONCLUSIONS: The urinary levels of podocyte-derived MPs were closely associated with podocyte injury and glomerulosclerosis, which could be useful for monitoring disease activity in CKD patients. Urinary podocyte-derived MPs might be a non-invasive biomarker for the evaluation of early CKD progression.

Heat Shock Cognate 70 Functions as A Chaperone for the Stability of Kinetochore Protein CENP-N in Holocentric Insect Silkworms.

The centromere, in which kinetochore proteins are assembled, plays an important role in the accurate congression and segregation of chromosomes during cell mitosis. Although the function of the centromere and kinetochore is conserved from monocentric to holocentric, the DNA sequences of the centromere and components of the kinetochore are varied among different species. Given the lack of core centromere protein A (CENP-A) and CENP-C in the lepidopteran silkworm Bombyx mori, which possesses holocentric chromosomes, here we investigated the role of CENP-N, another important member of the centromere protein family essential for kinetochore assembly. For the first time, cellular localization and RNA interference against CENP-N have confirmed its kinetochore function in silkworms. To gain further insights into the regulation of CENP-N in the centromere, we analyzed the affinity-purified complex of CENP-N by mass spectrometry and identified 142 interacting proteins. Among these factors, we found that the chaperone protein heat shock cognate 70 (HSC70) is able to regulate the stability of CENP-N by prohibiting ubiquitin-proteasome pathway, indicating that HSC70 could control cell cycle-regulated degradation of CENP-N at centromeres. Altogether, the present work will provide a novel clue to understand the regulatory mechanism for the kinetochore activity of CENP-N during the cell cycle.

A Novel LAMP Assay for the Detection of Phytophthora cinnamomi Utilizing a New Target Gene Identified From Genome Sequences.

Phytophthora cinnamomi is an ecologically and agriculturally significant plant pathogen. Early and accurate detection of P. cinnamomi is paramount to disease prevention and management. In this study, a loop-mediated isothermal amplification (LAMP) assay utilizing a new target gene Pcinn100006 identified from genomic sequence data was developed and evaluated for the detection of P. cinnamomi. This Pcinn100006 LAMP assay was found highly specific to P. cinnamomi. All 10 tested isolates of P. cinnamomi yielded positive results, whereas 50 isolates belonging to 16 other Phytophthora species, Globisporangium ultimum, and 14 fungal species lacked detection. This assay was 10 times more sensitive (100 pg in a 25-µl reaction mixture) than a conventional PCR assay (2 ng in a 50-µl reaction mixture) for detecting the genomic DNA of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, detection rates of P. cinnamomi using environmental DNAs extracted from 13 naturally infested rhizosphere samples were 100% in the Pcinn100006 LAMP assay versus 46% in the conventional PCR assay. Considering its higher accuracy and shorter time span, this Pcinn100006 LAMP assay is a promising diagnostic tool to replace conventional PCR-based and culture-dependent assays for screening of P. cinnamomi in regions at risk of infection or contamination.

Role of Podocyte Injury in Glomerulosclerosis.

Finding new therapeutic targets of glomerulosclerosis treatment is an ongoing quest. Due to a living environment of various stresses and pathological stimuli, podocytes are prone to injuries; moreover, as a cell without proliferative potential, loss of podocytes is vital in the pathogenesis of glomerulosclerosis. Thus, sufficient understanding of factors and underlying mechanisms of podocyte injury facilitates the advancement of treating and prevention of glomerulosclerosis. The clinical symptom of podocyte injury is proteinuria, sometimes with loss of kidney functions progressing to glomerulosclerosis. Injury-induced changes in podocyte physiology and function are actually not a simple passive process, but a complex interaction of proteins that comprise the anatomical structure of podocytes at molecular levels. This chapter lists several aspects of podocyte injuries along with potential mechanisms, including glucose and lipid metabolism disorder, hypertension, RAS activation, micro-inflammation, immune disorder, and other factors. These aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries.

Publisher Correction: Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy.

The REFERENCES 1-35 are wrong because of the error in the process of typesetting.

Platelet microparticles contribute to aortic vascular endothelial injury in diabetes via the mTORC1 pathway.

Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. The present study aimed to investigate the effects of PMPs in diabetes on aortic vascular endothelial injury and to explore the underlying mechanisms. Peritoneal injection of streptozotocin was used to generate a diabetic rat model in vivo, and human umbilical vein endothelial cells (HUVECs) treated with PMPs were used in vitro. PMP levels in the circulation and aorta tissues were time-dependently increased in streptozotocin-induced diabetic rats at weeks 4, 8, and 12 (P < 0.05). Aspirin significantly inhibited the PMP levels at each time point (P < 0.05). In diabetic rats, the endothelial nitric oxide levels were decreased significantly combined with increased endothelial permeability. PMPs were internalized by HUVECs and primarily accumulated around the nuclei. PMPs inhibited endothelial nitric oxide levels to about 50% and caused approximately twofold increase in reactive oxygen species production. Furthermore, PMPs significantly decreased the endothelial glycocalyx area and expression levels of glypican-1 and occludin (P < 0.05). Interestingly, the PMP-induced endothelial injuries were prevented by raptor siRNA and rapamycin. In conclusion, increased PMPs levels contribute to aortic vascular endothelial injuries in diabetes through activating the mTORC1 pathway.

ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis.

BACKGROUND: ANXA2 (Annexin A2) is a pleiotropic calcium-dependent phospholipid binding protein that is abnormally expressed in various cancers. We previously found that ANXA2 is upregulated in esophageal squamous cell carcinoma (ESCC). This study was designed to investigate the functional significance of ANXA2 dysregulation and underlying mechanism in ESCC. METHODS: Proliferation, migration, invasion and metastasis assay were performed to examine the functional roles of ANXA2 in ESCC cells in vitro and in vivo. Real-time RT-PCR, immunoblotting, ChIP, reporter assay, confocal-immunofluorescence staining, co-immunoprecipitation and ubiquitination assay were used to explore the molecular mechanism underlying the actions of deregulated ANXA2 in ESCC cells. RESULTS: Overexpression of ANXA2 promoted ESCC cells migration and invasion in vitro and metastasis in vivo through activation of the MYC-HIF1A-VEGF cascade. Notably, ANXA2 phosphorylation at Tyr23 by SRC led to its translocation into the nucleus and enhanced the metastatic potential of ESCC cells. Phosphorylated ANXA2 (Tyr23) interacted with MYC and inhibited ubiquitin-dependent proteasomal degradation of MYC protein. Accumulated MYC directly potentiated HIF1A transcription and then activated VEGF expression. Correlation between these molecules were also found in ESCC tissues. Moreover, dasatinib in combination with bevacizumab or ANXA2-siRNA produced potent inhibitory effects on the growth of ESCC xenograft tumors in vivo. CONCLUSIONS: This study provides evidence that highly expressed p-ANXA2 (Tyr23) contributes to ESCC progression by promoting migration, invasion and metastasis, and suggests that targeting the SRC-ANXA2-MYC-HIF1A-MYC axis may be an efficient strategy for ESCC treatment.

DNA methylation on N6-adenine in lepidopteran Bombyx mori.

Increasing knowledge of DNA methylation that occurs on the sixth position of adenine (N6-methyladenine, 6 mA) has emerged as a novel epigenetic mark in eukaryotes and plays an important role in regulating gene transcription, DNA replication and repair, transposable activities, and others. Here, we show DNA 6 mA methylation is present in Bombyx mori, a lepidopteran model insect, and identify the 6 mA methyltransferase, METTL4, and 6 mA demethylase, NMAD, which regulate the levels of 6 mA in embryogenesis and cultured cells of B. mori. Importantly, RNAi knockdown of METTL4 and NMAD not only induce cell cycle arrest at G1 phase but also result in defects of chromosome alignments at metaphase. We further demonstrate that 6 mA methylation is widely distributed across the genome of B. mori by 6 mA-Seq and primarily enriched in the regulatory regions as well as gene bodies. Integrated analysis of 6 mA-Seq and RNA-Seq reveals that 6 mA methylation in B. mori is preferentially related with lowly expressed genes and negatively correlated with active gene transcription, which provides a novel regulatory mechanism of DNA 6 mA methylation on target genes. Altogether, these data identify 6 mA methylation in B. mori and demonstrate a crucial role of 6 mA signaling in controlling cell cycle progression.

Inflammation-activated CXCL16 pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy.

Inflammation and lipid disorders play crucial roles in synergistically accelerating the progression of diabetic nephropathy (DN). In this study we investigated how inflammation and lipid disorders caused tubulointerstitial injury in DN in vivo and in vitro. Diabetic db/db mice were injected with 10% casein (0.5 mL, sc) every other day for 8 weeks to cause chronic inflammation. Compared with db/db mice, casein-injected db/db mice showed exacerbated tubulointerstitial injury, evidenced by increased secretion of extracellular matrix (ECM) and cholesterol accumulation in tubulointerstitium, which was accompanied by activation of the CXC chemokine ligand 16 (CXCL16) pathway. In the in vitro study, we treated HK-2 cells with IL-1ß (5 ng/mL) and high glucose (30 mmol/L). IL-1ß treatment increased cholesterol accumulation in HK-2 cells, leading to greatly increased ROS production, ECM protein expression levels, which was accompanied by the upregulated expression levels of proteins in the CXCL16 pathway. In contrast, after CXCL16 in HK-2 cells was knocked down by siRNA, the IL-1ß-deteriorated changes were attenuated. In conclusion, inflammation accelerates renal tubulointerstitial lesions in mouse DN via increasing the activity of CXCL16 pathway.

Combination of novel DR5 targeting agonistic scFv antibody TR2-3 with cisplatin shows enhanced synergistic antitumor activity in vitro and in vivo.

OBJECTIVES: To investigate the antitumor activity of a novel agonistic single chain fragment variable (scFv) antibody TR2-3 targeting death receptor 5 (DR5) combined with cisplatin in vitro and in vivo. METHODS: The in vitro cytotoxic effects of TR2-3 and cisplatin, alone or in combination on human cancer cell lines COLO205 and MDA-MB-231 were evaluated using the MTT assay. The apoptosis in cancer cells was evaluated by an Annexin V-PE apoptosis detection kit and flow cytometry. The mRNA and protein levels of DR5 were analyzed by real-time PCR and Western blot, respectively. Additionally, the in vivo antitumor activity of TR2-3 combined with cisplatin was evaluated in a xenograft model. RESULTS: The combination treatment with TR2-3 and cisplatin for 24 h on COLO205 and MDA-MB-231 cells showed significant cytotoxicity effects by MTT assay, compared with the alone treatment. Consistent with cell viability results, the cisplatin enhanced the apoptosis-inducing effects of TR2-3 in the COLO205 cells and MDA-MB-231 cells by flow cytometry. In addition, treatment with cisplatin alone for 24 h resulted in significantly up-regulating the mRNA and protein levels of DR5 in both COLO205 and MDA-MB-231 cell lines by q-PCR and Western blot assay. Moreover, the cytotoxic effects of TR2-3 can be blocked by adding the soluble DR5, and the blocking rate can be greatly reduced by co-treatment with cisplatin. These results indicated that cisplatin sensitized COLO205 and MDA-MB-231 cancer cells to TR2-3-mediated apoptosis by up-regulation of DR5 expression. Furthermore, combination therapy with TR2-3 and cisplatin enhanced tumor growth inhibition compared to treatment with TR2-3 or cisplatin alone in mice bearing COLO205 xenograft tumors. CONCLUSIONS: Our findings suggest that cisplatin enhanced the antitumor activity of TR2-3 in COLO205 and MDA-MB-231 cancer cells through up-regulation of DR5 expression. The TR2-3 combined with cisplatin may be a promising treatment for cancer therapy.