Med23 deficiency reprograms the tumor microenvironment to promote lung tumorigenesis.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. We previously found that Mediator complex subunit 23 (MED23) is important for the tumourigenicity of lung cancer cells with hyperactive Ras activity in vitro, although the in vivo function of MED23 in lung tumourigenesis remains to be explored. METHODS: In this study, we utilized well-characterized KrasG12D-driven non-small cell lung cancer mouse model to investigate the role of MED23 in lung cancer. The lung tumour progression was evaluated by H&E and IHC analysis. Western blotting and qRT-PCR assays were performed to detect changes in gene expression. Immune cells were analyzed by FACS technology. RNA-seq and reporter assays were conducted to explore the mechanism. RESULTS: We observed that lung epithelial Med23 deletion by adeno-Cre resulted in a significant increase in KrasG12D tumour number and size, which was further verified with another mouse model with Med23 specifically deleted in alveolar type II cells. Mice with lung-specific Med23 deficiency also exhibited accelerated tumourigenesis, and a higher proliferation rate for tumour cells, along with increased ERK phosphorylation. Notably, the numbers of infiltrating CD4+ T cells and CD8+ T cells were significantly reduced in the lungs of Med23-deficient mice, while the numbers of myeloid-derived suppressor cells (MDSCs) and Treg cells were significantly increased, suggesting the enhanced immune escape capability of the Med23-deficient lung tumours. Transcriptomic analysis revealed that the downregulated genes in Med23-deficient lung tumour tissues were associated with the immune response. Specifically, Med23 deficiency may compromise the MHC-I complex formation, partially through down-regulating B2m expression. CONCLUSIONS: Collectively, these findings revealed that MED23 may negatively regulate Kras-induced lung tumourigenesis in vivo, which would improve the precise classification of KRAS-mutant lung cancer patients and provide new insights for clinical interventions.

Microenvironment modulation by key regulators of RNA N6-methyladenosine modification in respiratory allergic diseases.

BACKGROUND: RNA N6-methyladenosine (m6A) regulators are considered post-transcriptional regulators that affect several biological functions, and their role in immunity, in particular, is emerging. However, the role of m6A regulators in respiratory allergic diseases remains unclear. Therefore, we aimed to investigate the role of key m6A regulators in mediating respiratory allergic diseases and immune microenvironment infiltration characteristics. METHODS: We downloaded gene expression profiles of respiratory allergies from the Gene Expression Omnibus (GEO) database and we performed hierarchical clustering, difference analysis, and construction of predictive models to identify hub m6A regulators that affect respiratory allergies. Next, we investigate the underlying biological mechanisms of key m6A regulators by performing PPI network analysis, functional enrichment analysis, and immune microenvironment infiltration analysis. In addition, we performed a drug sensitivity analysis on the key m6A regulator, hoping to be able to provide some implications for clinical medication. RESULTS: In this study, we identified four hub m6A regulators that affect the respiratory allergy and investigated the underlying biological mechanisms. In addition, studies on the characteristics of immune microenvironment infiltration revealed that the expression of METTL14, METTL16, and RBM15B correlated with the infiltration of the mast and Th2 cells in respiratory allergy, and METTL16 expression was found to be significantly negatively correlated with macrophages for the first time (R = -0.53, P < 0.01). Finally, a key m6A regulator, METTL14, was screened by combining multiple algorithms. In addition, by performing a drug sensitivity analysis on METTL14, we hypothesized that it may play an important role in the improvement of allergic symptoms in the upper and lower airways with topical nasal glucocorticoids. CONCLUSIONS: Our findings suggest that m6A regulators, particularly METTL14, play a crucial role in the development of respiratory allergic diseases and the infiltration of immune cells. These results may provide insight into the mechanism of action of methylprednisolone in treating respiratory allergic diseases.

Discovery of novel 2-aminopyridine derivatives as ROS1 and ALK dual inhibitors to combat drug-resistant mutants including ROS1G2032R and ALKG1202R.

Clinical treatment by FDA-approved ROS1/ALK inhibitor Crizotinib significantly improved the therapeutic outcomes. However, the emergence of drug resistance, especially driven by acquired mutations, have become an inevitable problem and worsened the clinical effects of Crizotinib. To combat drug resistance, some novel 2-aminopyridine derivatives were designed rationally based on molecular simulation, then synthesised and subjected to biological test. The preferred spiro derivative C01 exhibited remarkable activity against CD74-ROS1G2032R cell with an IC50 value of 42.3 nM, which was about 30-fold more potent than Crizotinib. Moreover, C01 also potently inhibited enzymatic activity against clinically Crizotinib-resistant ALKG1202R, harbouring a 10-fold potency superior to Crizotinib. Furthermore, molecular dynamic disclosed that introducing the spiro group could reduce the steric hindrance with bulky side chain (Arginine) in solvent region of ROS1G2032R, which explained the sensitivity of C01 to drug-resistant mutant. These results indicated a path forward for the generation of anti Crizotinib-resistant ROS1/ALK dual inhibitors.

A Novel and Highly Selective Epidermal Growth Factor Receptor Inhibitor, SMUZ106, for the Treatment of Glioblastoma.

Targeting the epidermal growth factor receptor (EGFR) is one of the potential ways to treat glioblastoma (GBM). In this study, we investigate the anti-GBM tumor effects of the EGFR inhibitor SMUZ106 in both in vitro and in vivo conditions. The effects of SMUZ106 on the growth and proliferation of GBM cells were explored through MTT and clone formation experiments. Additionally, flow cytometry experiments were conducted to study the effects of SMUZ106 on the cell cycle and apoptosis of GBM cells. The inhibitory activity and selectivity of SMUZ106 to the EGFR protein were proved by Western blotting, molecular docking, and kinase spectrum screening methods. We also conducted a pharmacokinetic analysis of SMUZ106 hydrochloride following i.v. or p.o. administration to mice and assessed the acute toxicity level of SMUZ106 hydrochloride following p.o. administration to mice. Subcutaneous and orthotopic xenograft models of U87MG-EGFRvIII cells were established to assess the antitumor activity of SMUZ106 hydrochloride in vivo. SMUZ106 could inhibit the growth and proliferation of GBM cells, especially for the U87MG-EGFRvIII cells with a mean IC50 value of 4.36 µM. Western blotting analyses showed that compound SMUZ106 inhibits the level of EGFR phosphorylation in GBM cells. It was also shown that SMUZ106 targets EGFR and presents high selectivity. In vivo, the absolute bioavailability of SMUZ106 hydrochloride was 51.97%, and its LD50 exceeded 5000 mg/kg. SMUZ106 hydrochloride significantly inhibited GBM growth in vivo. Furthermore, SMUZ106 inhibited the activity of U87MG-resistant cells induced by temozolomide (TMZ) (IC50: 7.86 µM). These results suggest that SMUZ106 hydrochloride has the potential to be used as a treatment method for GBM as an EGFR inhibitor.

Association of CYP2C19, CYP3A4 and ABCC2 polymorphisms and voriconazole plasma concentrations in Uygur pediatric patients.

Aim: To evaluate the association between CYP2C19, CYP3A4 and ABCC2 polymorphisms and voriconazole plasma concentrations in Uygur pediatric patients with allogeneic hematopoietic stem cell transplantation. Materials & methods: High performance liquid chromatography-mass spectrometry was employed to monitor voriconazole concentrations. First-generation sequencing was performed to detect gene polymorphisms. Results: Voriconazole concentrations of normal metabolizers were significantly higher than those of intermediate (p < 0.05) and ultrafast (p < 0.001) metabolizers. Patients with ABCC2 GG and GA genotypes exhibited significantly lower voriconazole concentrations compared with patients with the AA genotype (p < 0.05). Conclusion: These results demonstrate a significant association between voriconazole concentrations and the CYP2C19 phenotype in Uygur pediatric patients with allogeneic hematopoietic stem cell transplantation.

Deficiency of astrocyte CysLT1R ameliorates depression-like behaviors in mice by modulating glutamate synaptic transmission.

Our previous study suggests that hippocampal cysteinyl leukotriene receptor 1 (CysLT1R) could be involved in depression. Herein we hypothesize that CysLT1R may regulate depression by affecting synaptic glutamate cycling based on existence of CysLT1R in the astrocytes that participate in occurrence of depression. We found that CysLT1R expression was significantly increased in the astrocyte of chronic unpredictable mild stress (CUMS)-induced depression-like mice, CysLT1R astrocyte-specific conditional knockout (AcKO) significantly improved depression-like behaviors, as indicated by decreased immobility time in the forced swimming test and tail suspension test and increased sucrose preference in the sucrose preference test, and knockdown of CysLT1R in the astrocyte of dentate gyrus (DG), the region with the most significant increase of CysLT1R in the astrocyte of depression-like mice, produced similar effects. Correspondingly, overexpression of CysLT1R in the astrocyte of DG induced depression-like behaviors in mice. The further study showed that CysLT1R AcKO ameliorated synaptic plasticity impairment, as reflected by increased synapse, LTP and PSD95, and promoted glutamate transporter 1 (GLT-1) expression by inhibiting NF-κB p65 nuclear translocation mediated by ß-arestin2 and clatrhin, subsequently decreased glutamate in synaptic cleft and GluN2B on postsynaptic membrane in depression-like mice. The present study also showed that GLT-1 agonist or NF-κB inhibitor ameliorated depressive-like behaviors induced by overexpression of the astrocyte CysLT1R of DG. Our study demonstrated that astrocyte CysLT1R regulated depression by modulating glutamate synaptic transmission, suggesting that CysLT1R could be a potential target for developing novel drugs of anti-depression.

CD169+ subcapsular sinus macrophage-derived microvesicles are associated with light zone follicular dendritic cells.

Follicular dendritic cells (FDCs) are a specialized type of stromal cells that exclusively reside in B-cell follicles. When inflammation occurs, the FDC network is reorganized to support germinal center (GC) polarization into the light zone (LZ) and dark zone (DZ). Despite the indispensable role of FDCs in supporting humoral responses, the FDC regulatory requirements remain incompletely defined. In this study, we unexpectedly observed an accumulation of CD169+ subcapsular sinus macrophage (SSM)-derived microvesicles (MVs) in the B-cell zone, which were tightly associated with the FDC network. Interestingly, a selective deposition of CD169+ MVs was detected in both GC LZ FDCs in secondary follicles and on predetermined LZ FDCs in primary follicles. The ablation of CD169+ MVs, resulting from SSM depletion, resulted in significantly decreased expression of LZ-related genes in FDCs. In addition, we found that CD169+ MVs could colocalize with fluorescently tagged antigen-containing immune complexes (ICs), supporting a possible role of CD169+ MVs in transporting antigens to the FDC network. Thus, our data reveal intimate crosstalk between FDCs and SSMs located outside B-cell follicles via SSM-released MVs, providing a novel perspective on the mechanisms underlying the regulation of FDC maturation and polarization.

Modified Nuss procedure with a novel steel bar in patients with pectus excavatum post-congenital heart surgery.

OBJECTIVES: Pectus excavatum (PE) can be secondary in patients who underwent sternotomy for cardiac surgery. Retrosternal adhesions increase the complexity and risk of traditional Nuss repair. Thus, we summarized the outcomes of our modified Nuss procedure using a newly designed bar. METHODS: A retrospective analysis was performed on 35 patients who underwent modified PE repair after open heart surgery from January 2011 to July 2019. The surgery was performed using a novel bar with no need for intraoperative reshaping and rotation, assisted by thoracoscopy and subxiphoid incision when necessary. RESULTS: There were 19 males and 16 females with a median age of 5.3 years (interquartile range, 4.1-10.9) at PE repair. All patients underwent the modified procedure uneventfully with no death. The median operating time was 70 min. Twenty-nine (82.9%) patients required subxiphoid incision assistance. There was 1 case (2.8%) with unexpected sternotomy due to intraoperative bleeding. The median length of postoperative hospital stay was 4 days. During the median 3.5 years of follow-up, no bar dislocation was found and 30 (85.7%) patients had their bars removed with no recurrence recorded. After PE repair, the Haller index improved significantly (2.6 ± 0.4 vs 4.9 ± 1.3, P < 0.05) and further decreased till the time of bar removal (2.5 ± 0.4 vs 2.6 ± 0.4, P < 0.05). All patients were satisfied with the cosmetic outcome. CONCLUSIONS: The novel bar can be placed and removed easily with a low rate of adverse events. This modified Nuss procedure seems to be a safe, effective and convenient approach for the management of PE after cardiac surgery.

Oriented Localization of Surgical Tools by Location Encoding.

Surgical tool localization is the foundation to a series of advanced surgical functions e.g. image guided surgical navigation. For precise scenarios like surgical tool localization, sophisticated tools and sensitive tissues can be quite close. This requires a higher localization accuracy than general object localization. And it is also meaningful to know the orientation of tools. To achieve these, this paper proposes a Compressive Sensing based Location Encoding scheme, which formulates the task of surgical tool localization in pixel space into a task of vector regression in encoding space. Furthermore with this scheme, the method is able to capture orientation of surgical tools rather than simply outputting horizontal bounding boxes. To prevent gradient vanishing, a novel back-propagation rule for sparse reconstruction is derived. The back-propagation rule is applicable to different implementations of sparse reconstruction and renders the entire network end-to-end trainable. Finally, the proposed approach gives more accurate bounding boxes as well as capturing the orientation of tools, and achieves state-of-the-art performance compared with 9 competitive both oriented and non-oriented localization methods on a mainstream surgical image dataset: m2cai16-tool-locations. A range of experiments support our claim that regression in CSLE space performs better than traditionally detecting bounding boxes in pixel space.

Long non­coding RNA fer­1­like family member 4 serves as a tumor suppressor in laryngeal squamous cell carcinoma cells via regulating the AKT/ERK signaling pathway.

Laryngeal squamous cell carcinoma (LSCC) is a common type of malignant tumor of the head and neck. An increasing number of studies have illustrated that long non­coding RNAs (lncRNAs) serve an important role in the occurrence and development of LSCC. Therefore, the present study aimed to investigate the expression changes and mechanism of lncRNA fer­1­like family member 4 (FER1L4) in the progression of LSCC. The expression levels of FER1L4 in LSCC cell lines (AMC­HN­8, Tu 686, M4E and M2E) and a normal cell line (HBE135­E6E7) were analyzed using reverse transcription­quantitative PCR. The FER1L4 overexpression plasmid (plasmid­FER1L4) was subsequently transfected into Tu 686 cells to upregulate the expression levels of FER1L4. Cell viability was detected using a Cell Counting Kit­8 assay, cell proliferation was analyzed using a colony formation assay, apoptosis was examined by flow cytometry, and cell migration and invasion were determined using wound healing and Transwell assays, respectively. In addition, the plasmid­FER1L4 cells were also treated with insulin­like growth factor 1 (IGF­1) to determine the effect of FER1L4 on the AKT/ERK signaling pathway, and the effect of the plasmid­FER1L4 on the expression levels of AKT/ERK signaling pathway­related proteins were analyzed using western blotting. The results of the present study revealed that FER1L4 expression levels were downregulated in AMC­HN­8 and Tu 686 cells. Notably, FER1L overexpression significantly reduced the cell viability, proliferation, migration and invasion of LSCC cells, while promoting apoptosis. Meanwhile, the plasmid­FER1L4 also significantly suppressed the phosphorylation levels of AKT and ERK. Further studies indicated that the aforementioned changes could be reversed by IGF­1, indicating FER1L4 may regulate the progression of LSCC cells by inhibiting the AKT/ERK signaling pathway. In conclusion, the present study provided a potential novel direction for the treatment of LSCC in the future and suggested that FER1L4 may be a new target in this field.

The cytochrome P450 metabolic profiling of SMU-B in vitro, a novel small molecule tyrosine kinase inhibitor.

A novel small molecule tyrosine kinase inhibitor 6-[6-Amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-3-pyridyl]-1'-methylspiro[indoline-3,4'-piperidine]-2-one (SMU-B) had good activity against ALK (anaplastic lymphoma kinase) and ROS1 (c-ros oncogene 1) targets in non-small-cell lung cancer. The excellent bioactivity of SMU-B highlights the importance of determining its metabolic traits, which could provide meaningful information for further pharmacokinetic studies of SMU-B. In this work, we studied the metabolism of SMU-B in human liver microsomes. Three metabolites of SMU-B were identified by a quadrupole-time of flight tandem mass spectrometer (Q-TOF-MS), and the metabolic pathways of SMU-B were demethylation, dehydrogenation and oxidation. CYP3A4/5 was the principal isoform involved in SMU-B metabolism, as shown by chemical inhibition and recombination human enzyme studies. Additionally, a predication with a molecular docking model confirmed that SMU-B could interact with the active sites of CYP3A4 and CYP3A5. This study illuminates the metabolic profile of the anti-tumor drug SMU-B, which will accelerate its clinical use.

Identification of dysregulated serum miR-508-3p and miR-885-5p as potential diagnostic biomarkers of clear cell renal carcinoma.

Clear cell renal cell carcinoma (ccRCC), the most common subtype, accounts for approximately 80% of all RCC cases. ccRCC patients typically present with an advanced stage at the time of diagnosis resulting in a poor patient prognosis. The present study aimed to identify novel potential microRNAs (miRNAs or miRs) in peripheral blood as biomarkers for the detection of ccRCC. Candidate miRNAs were selected through integrated analysis of the Gene Expression Omnibus (GEO) database, The Cancer Genome Atlas (TCGA) database, and from clinical samples. The expression levels of miRNAs were quantified using reverse transcription­quantitative PCR. Receiver operating characteristic (ROC) curve analysis was used to explore the diagnostic values of the miRNAs. Bioinformatic analysis of candidate miRNAs was conducted by using the STRING database. After an integrated analysis of the GEO and TCGA databases, four miRNAs were found to be consistently dysregulated in ccRCC tissues. Then, their expression levels in serum and diagnostic utilities were further explored. We discovered that serum miR­508­3p and miR­885­5p were significantly dysregulated in ccRCC patients with marked diagnostic values. The area under the ROC curve (AUC) of serum miR­508­3p and miR­885­5p was 0.80 (95% CI, 0.73­0.87) and 0.87 (95% CI, 0.79­0.95), respectively. Functional enrichment analysis revealed that both miR­508­3p and miR­885­5p were closely associated with cellular metabolic processes. In conclusion, serum miR­508­3p and miR­885­5p are novel potential biomarkers for the diagnosis of ccRCC.

Design, synthesis and biological evaluations of 2-amino-4-(1-piperidine) pyridine derivatives as novel anti crizotinib-resistant ALK/ROS1 dual inhibitors.

ALK and ROS1 kinases have become promising therapeutic targets since Crizotinib was used to treat non-small-cell lung cancer clinically. Aiming to explore new potent inhibitors, a series of 2-amino-4-(1-piperidine) pyridine derivatives that stabilized a novel DFG-shifted conformation in the kinase domain of ALK were designed and synthesized on the base of lead compound A. Biological evaluation highlighted that most of these new compounds could also potently inhibit ROS1 kinase, leading to the promising inhibitors against both ROS1 and ALK. Among them, the representative compound 2e stood out potent anti-proliferative activity against ALK-addicted H3122 and ROS1-addicted HCC78 cell lines (IC50 = 6.27 µM and 10.71 µM, respectively), which were comparable to that of Crizotinib. Moreover, 2e showed impressive enzyme activity against clinically Crizotinib-resistant ALKL1196M with an IC50 value of 41.3 nM, which was about 2-fold more potent than that of Crizotinib. 2e also showed potent inhibitory activity in about 6-fold superior to Crizotinib (IC50: 104.7 nM vs. 643.5 nM) in Ba/F3 cell line harboring ROS1G2032R. Furthermore, molecular modeling disclosed that all the representative inhibitors could dock into the active site of ALK and ROS1, which gave a probable explanation of anti Crizotinib-resistant mutants. These results indicated that our work has established a path forward for the generation of anti Crizotinib-resistant ALK/ROS1 dual inhibitors.

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets.

Rodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first-phase glucose-stimulated insulin secretion (GSIS) compared with cultured-intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first-phase GSIS. Lastly, the lentiviral-mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.

Evolution strategy of ROS1 kinase inhibitors for use in cancer therapy.

The abnormal expression of c-ros oncogene1 receptor tyrosine kinase (ROS1) has been identified as clinically actionable oncogenic driver in non-small-cell lung cancer. Since crizotinib was approved by the US FDA for the treatment of advanced ROS1-positive non-small-cell lung cancer, ROS1 kinase has become a promising therapeutic target. Under the guidance of some advanced computer-assisted technologies, such as structure-based drug design, homology modeling and lipophilic efficiency parameters, several potent and selective inhibitors against wild-type and mutant ROS1 were designed and synthesized. In this article, we will review a series of scaffolds targeting ROS1 kinase from the hit-to-drug evolution strategies of their representative compounds and it is hoped that these design strategies would facilitate medicinal chemists to optimize the process of drug design.

[Screening active ingredients of Shenkangwan that regulate endothelial-mesenchymal transition of endothelial cells in vitro].

OBJECTIVE: To screen the effective components of Shenkangwan that regulate endothelial-mesenchymal transition in endothelial cells for optimizing prescription of Shenkangwan. METHODS: ALK5 was identified as one of the target receptors that regulate endothelial-mesenchymal transition of endothelial cells using molecular docking technique. Nine molecules were screened as the candidate effective components in Shenkangwan, among which calycosin, ononin and stigmasterol were selected for testing. Glomerular epithelial cells were exposed to high glucose and treated with calycosin, ononin, or stigmasterol, and the cellular expressions of α-smooth muscle actin (α-SMA) and vimentin mRNA were detected with real-time fluorescence quantitative PCR. The phosphorylation of SMAD2/3 in the cells was detected using Western blotting. RESULTS: Calycosin, ononin and stigmasterol did not produce significant cytotoxicity in glomerular epithelial cells (P>0.05). The cells exposed to high glucose and calycosin treatment showed significantly decreased mRNA levels of α-SMA and vimentin (P<0.05) and inhibited phosphorylation of SMAD2/3. Ononin and stigmasterol did not produce such effects in the cells. CONCLUSION: In endothelial cells with high glucose-induced injury, calycosin can inhibit the up-regulation of α-SMA and vimentin and inhibit phosphorylation of SMAD2/3 to regulate endothelial-mesenchymal transition and improve diabetic nephropathy.

Bit-based support vector machine nonlinear detector for millimeter-wave radio-over-fiber mobile fronthaul systems.

An effective bit-based support vector machine (SVM) is proposed as a non-parameter nonlinear mitigation approach in the millimeter-wave radio-over-fiber (RoF) mobile fronthaul (MFH) system for various modulation formats. First, we analyze the impairments originated from nonlinearities in the millimeter-wave RoF system. Then we introduce the operation principle of the bit-based SVM detector. As a classifier, the SVM can create nonlinear decision boundaries by kernel function to mitigate the distortions caused by both linear and nonlinear noise. In our design, SVM can learn and capture the link characteristics from only a few training data without requiring the prior estimation of the system link. The bit-based SVM only needs log2M SVMs to detect the signal of M-order modulation format. Experimental results have been obtained to verify the feasibility of the proposed method. The sensitivities are improved by 1.2-dB for 16-QAM, 1.3-dB for 64-QAM, 1.8-dB for 16-APSK and 1.3-dB for 32-APSK at BER = 1E-3 with SVM detector, respectively. The proposed bit-based SVM gains a large improvement in the nonlinear system tolerance and outperforms the system employing k-means algorithm.

Lipid-based DNA/siRNA transfection agents disrupt neuronal bioenergetics and mitophagy.

A multitude of natural and artificial compounds have been recognized to modulate autophagy, providing direct or, through associated pathways, indirect entry points to activation and inhibition. While these pharmacological tools are extremely useful in the study of autophagy, their abundance also suggests the potential presence of unidentified autophagic modulators that may interfere with experimental designs if applied unknowingly. Here, we report unanticipated effects on autophagy and bioenergetics in neuronal progenitor cells (NPCs) incubated with the widely used lipid-based transfection reagent lipofectamine (LF), which induced mitochondria depolarization followed by disruption of electron transport. When NPCs were exposed to LF for 5 h followed by 24, 48, and 72 h in LF-free media, an immediate increase in mitochondrial ROS production and nitrotyrosine formation was observed. These events were accompanied by disrupted mitophagy (accumulation of dysfunctional and damaged mitochondria, and of LC3II and p62), in an mTOR- and AMPK-independent manner, and despite the increased mitochondrial PINK1 (PTEN-inducible kinase 1) localization. Evidence supported a role for a p53-mediated abrogation of parkin translocation and/or abrogation of membrane fusion between autophagosome and lysosomes. While most of the outcomes were LF-specific, only two were shared by OptiMEM exposure (with no serum and reduced glucose levels) albeit at lower extents. Taken together, our findings show that the use of transfection reagents requires critical evaluation with respect to consequences for overall cellular health, particularly in experiments designed to address autophagy-inducing effects and/or energy stress.

Altered redox mitochondrial biology in the neurodegenerative disorder fragile X-tremor/ataxia syndrome: use of antioxidants in precision medicine.

A 55-200 expansion of the CGG nucleotide repeat in the 5'-UTR of the fragile X mental retardation 1 gene (FMR1) is the hallmark of the triplet nucleotide disease known as the "premutation" as opposed to those with >200 repeats, known as the full mutation or fragile X syndrome. Originally, premutation carriers were thought to be free of phenotypic traits; however, some are diagnosed with emotional and neurocognitive issues and, later in life, with the neurodegenerative disease fragile X-associated tremor/ataxia syndrome (FXTAS). Considering that mitochondrial dysfunction has been observed in fibroblasts and post-mortem brain samples from carriers of the premutation, we hypothesized that mitochondrial dysfunction-derived ROS may result in cumulative oxidative-nitrative damage. Fibroblasts from premutation carriers (n=31, all FXTAS-free except 8), compared to age- and sex-matched controls (n=25), showed increased mitochondrial ROS production, impaired Complex I activity, lower expression of MIA40 (rate-limiting step of the redox-regulated mitochondrial-disulfide-relay-system), increased mtDNA deletions, and increased biomarkers of lipid and protein oxidative-nitrative damage. Most of the outcomes were more pronounced in FXTAS-affected individuals. Significant recovery of mitochondrial mass and/or function was obtained with superoxide or hydroxyl radicals' scavengers, a glutathione peroxidase analog, or by overexpressing MIA40. The effects of ethanol (a hydroxyl radical scavenger) were deleterious, while others (by N-acetyl-cysteine, quercetin and epigallocatechin-3-gallate) were outcome- and/or carrier-specifics. The use of antioxidants in the context of precision medicine is discussed with the goal of improving mitochondrial function in carriers with the potential of decreasing the morbidity and/or delaying FXTAS onset.