Effect of RPL27 knockdown on the proliferation and apoptosis of human liver cancer cells.

RPL27 is linked to the development of various diseases including malignant tumors. RPL27 may play an oncogenic function in hepatocellular carcinoma (HCC), but this is unknown. So, the aim of this study was to investigate how the human liver cancer cell lines SNU449 and HepG2 responded to RPL27 knockdown in terms of proliferation and apoptosis. SNU449 and HepG2 were cultured and infected with shCon and shRPL27 lentiviral particles to induce RPL27 knockdown, and then RPL27 expression was detected using qPCR and Western blot. Cell proliferation was measured using CCK8, cell cloning, cell scraping, and transwell migration and invasion, while apoptosis was measured using flow cytometry (FCM). The qPCR revealed that mRNA expression of RPL27 decreased after knocking down RPL27 in cells. The CCK8 and cell cloning assay confirmed that knocking down RPL27 significantly reduced cell viability. The cell scratch assay and transwell assays showed that the proliferation rate decreased after knocking down RPL27. A substantial increase in apoptotic cells was discovered by FCM. According to WB, RPL27 knockdown increased the expression of Bax and Caspase-3 while decreasing the expression of bcl-2. The findings showed that RPL27 knockdown inhibited cell proliferation in SNU449 and HepG2 via inducing apoptosis, proving that RPL27 is a novel gene linked with HCC and is crucial for both proliferation and apoptosis. These outcomes imply that RPL27 may be a potential target for liver cancer diagnosis and therapy.

Mutated Flt3Lg Provides Reduced Flt3 Recycling Compared to Wild-Type Flt3Lg and Retains the Specificity of Flt3Lg-Based CAR T-Cell Targeting in AML Models.

The cells of acute myeloid leukemia are defined by clonal growth and heterogenous immunophenotypes. Chimeric antigen receptors (CARs) commonly recognize molecular targets by single-chain antibody fragments (scFvs) specific to a tumor-associated antigen. However, ScFvs may form aggregates, thus stimulating tonic CAR T-cell activation and reducing CAR T-cell functioning in vivo. Harnessing natural ligands as recognition parts of CARs, specific targeting of membrane receptors can be achieved. Previously, we presented ligand-based Flt3-CAR T-cells targeting the Flt3 receptor. The extracellular part of Flt3-CAR consisted of full-size Flt3Lg. Meanwhile, upon recognition, Flt3-CAR may potentially activate Flt3, triggering proliferative signaling in blast cells. Moreover, the long-lasting presence of Flt3Lg may lead to Flt3 downregulation. In this paper, we present mutated Flt3Lg-based Flt3m-CAR ('m'-for 'mutant') T-cells targeting Flt3. The extracellular part of Flt3m-CAR consists of full-length Flt3Lg-L27P. We have determined that ED50 for recombinant Flt3Lg-L27P produced in CHO cells is at least 10-fold higher than for the wild-type Flt3Lg. We show that the mutation in the recognizing domain of Flt3m-CAR did not affect the specificity of Flt3m-CAR T-cells when compared to Flt3-CAR T-cells. Flt3m-CAR T-cells combine the specificity of ligand-receptor recognition with reduced Flt3Lg-L27P bioactivity, leading to potentially safer immunotherapy.

The loss of DLG2 isoform 7/8, but not isoform 2, is critical in advanced staged neuroblastoma.

BACKGROUND: Neuroblastoma is a childhood neural crest tumor showing large clinical and genetic heterogeneity, one form displaying 11q-deletion is very aggressive. It has been shown that 11q-deletion results in decreased expression of DLG2, a gene residing in the deleted region. DLG2 has a number of different isoforms with the main difference is the presence or absence of a L27 domain. The L27 domain containing DLG proteins can form complexes with CASK/MPP and LIN7 protein family members, which will control cell polarity and signaling. METHODS: We evaluated the DLG gene family and the LIN7 gene family for their expression in differently INSS staged neuroblastoma from publically available data and primary tumors, we included two distinct DLG1 and DLG2 N-terminal transcript isoforms encoding L27 domains for their expression. Functionality of DLG2 isoforms and of LIN7A were evaluated in the 11q-deleted neuroblastoma cell line SKNAS. RESULTS: In neuroblastoma only two DLG2 isoforms were expressed: isoform 2 and isoform 7/8. Using the array data we could determine that higher expression of DLG members that contain L27 domains correlated to better survival and prognosis. Whilst DLG1 showed a decrease in both isoforms with increased INSS stage, only the full length L27 containing DLG2 transcripts DLG2-isoform 7/8 showed a decrease in expression in high stage neuroblastoma. We could show that the protein encoded by DLG2-isoform 7 could bind to LIN7A, and increased DLG2-isoform 7 gene expression increased the expression of LIN7A, this reduced neuroblastoma cell proliferation and viability, with increased BAX/BCL2 ratio indicating increased apoptosis. CONCLUSION: We have provided evidence that gene expression of the L27 domain containing DLG2-isoform 7/8 but not L27 domain lacking DLG2-isoform 2 is disrupted in neuroblastoma, in particular in the aggressive subsets of tumors. The presence of the complete L27 domain allows for the binding to LIN7A, which will control cell polarity and signaling, thus affecting cancer cell viability.

Identification and characterization of SaRpAMP, a 60S ribosomal protein L27-derived antimicrobial peptide from amur catfish, Silurus asotus.

Aquatic freshwater fish like catfish, Silurus asotus, lives in microbe-rich environments, which enable this fish to develop necessary defense mechanisms. Antimicrobial peptides, along with other innate immune factors, are regarded as an important group in this defense. An antimicrobial peptide, which was isolated from the skin of S. asotus, was identified as a C-terminal fragment of 60S ribosomal protein L27 from S. asotus. The peptide was, then, designated Silurus asotus 60S ribosomal protein L27-derived antimicrobial peptide, SaRpAMP. Primary structure analyses and cDNA cloning revealed that SaRpAMP was 4185.36 Da and composed of 33 amino acids (AAs). Its precursor had a total of 136 AAs containing a pro-sequence of 103 AAs encoded by the nucleotide sequence of 512 bp that comprises a 5' untranslated region (UTR) of 32 bp, an open reading frame (ORF) of 411 bp, and a 3' UTR of 69 bp. Secondary structure analyses showed that SaRpAMP had two α-helices with turns and coils and an amphiphilic structure, a finding consistent with the 3D model of the peptide. SaRpAMP exhibited potent antibacterial activity comparable to piscidin 1, a powerful positive control. Its antimicrobial activity against fungus C. albicans was relatively weak. The antimicrobial activity of SaRpAMP was not diminished by heat treatment and changes in pH but was abolished by proteolytic enzyme digestion. Membrane permeability assays suggested that SaRpAMP interacts with both the outer and inner bacterial membranes. This was consistent with the results of lipid titration and quenching of Trp fluorescence that demonstrated SaRpAMP's interaction with acidic liposomes. Collectively, these findings suggest that the identified peptide, SaRpAMP, was the first antimicrobial peptide reported to be derived from the C-terminal region of 60S ribosomal protein L27. The findings also suggest that the action mechanism of SaRpAMP involved the interaction of the peptide with the bacterial membranes.

The multi-PDZ domain protein-1 (MUPP-1) expression regulates cellular levels of the PALS-1/PATJ polarity complex.

MUPP-1 (multi-PDZ domain protein-1) and PATJ (PALS-1-associated tight junction protein) proteins are closely related scaffold proteins and bind to many common interactors including PALS-1 (protein associated with Lin seven) a member of the Crumbs complex. Our goal is to understand how MUPP-1 and PATJ and their interaction with PALS-1 are regulated in the same cells. We have shown that in MCF10A cells there are at least two different and co-existing complexes, PALS-1/MUPP-1 and PALS-1/PATJ. Surprisingly, MUPP-1 levels inversely correlated with PATJ protein levels by acting on the stabilization of the PATJ/PALS-1 complex. Upon MUPP-1 depletion, the increased amounts of PATJ are in part localized at the migrating front of MCF10A cells and are able to recruit more PAR3 (partition defective 3). All together these data indicate that a precise balance between MUPP-1 and PATJ is achieved in epithelial cells by regulating their association with PALS-1.

RPL27 contributes to colorectal cancer proliferation and stemness via PLK1 signaling.

Although expression of ribosomal protein L27 (RPL27) is upregulated in clinical colorectal cancer (CRC) tissue, to the best of our knowledge, the oncogenic role of RPL27 has not yet been defined. The present study aimed to investigate whether targeting RPL27 could alter CRC progression and determine whether RPL27 gains an extra­ribosomal function during CRC development. Human CRC cell lines HCT116 and HT29 were transfected with RPL27­specific small interfering RNA and proliferation was assessed in vitro and in vivo using proliferation assays, fluorescence­activated cell sorting (FACS) and a xenograft mouse model. Furthermore, RNA sequencing, bioinformatic analysis and western blotting were conducted to explore the underlying mechanisms responsible for RPL27 silencing­induced CRC phenotypical changes. Inhibiting RPL27 expression suppressed CRC cell proliferation and cell cycle progression and induced apoptotic cell death. Targeting RPL27 significantly inhibited growth of human CRC xenografts in nude mice. Notably, polo­like kinase 1 (PLK1), which serves an important role in mitotic cell cycle progression and stemness, was downregulated in both HCT116 and HT29 cells following RPL27 silencing. RPL27 silencing reduced the levels of PLK1 protein and G2/M­associated regulators such as phosphorylated cell division cycle 25C, CDK1 and cyclin B1. Silencing of RPL27 reduced the migration and invasion abilities and sphere­forming capacity of the parental CRC cell population. In terms of phenotypical changes in cancer stem cells (CSCs), RPL27 silencing suppressed the sphere­forming capacity of the isolated CD133+ CSC population, which was accompanied by decreased CD133 and PLK1 levels. Taken together, these findings indicated that RPL27 contributed to the promotion of CRC proliferation and stemness via PLK1 signaling and RPL27 may be a useful target in a next­generation therapeutic strategy for both primary CRC treatment and metastasis prevention.

The N-terminal region of yeast mitoribosomal Mrp7/bL27m protein serves to optimize translation of nascent chains competent for OXPHOS complex assembly.

The extreme N-terminal residues of the mitochondrial ribosomal bL27m proteins reside within the ribosomal peptidyl transferase center (PTC) and are conserved from their bacterial ancestors. Mutation or truncation of the N-terminal region of the yeast Mrp7/bL27m protein did not inhibit protein synthesis but significantly impacted the efficacy of the mitochondrial translational process with respect to yielding proteins competent to assemble into functional oxidative phosphorylation enzymes. The requirement for the N-terminal residues of Mrp7/bL27m to support normal mitotranslation was more apparent under respiratory growth. We demonstrate that the N-terminal region of Mrp7/bL27m impacts the environment of the PTC and speculate the bL27m proteins serve to fine-tune and optimize mitoribosomal activity with respect to the downstream fate of the nascent chain.

Optimization of Process Parameters in CNC Turning of Aluminum 7075 Alloy Using L27 Array-Based Taguchi Method.

With the advent of the industrial revolution 4.0, the goal of the manufacturing industry is to produce a large number of products in relatively less time. This study applies the Taguchi L27 orthogonal array methodological paradigm along with response surface design. This work optimizes the process parameters in the turning of Aluminum Alloy 7075 using a Computer Numerical Control (CNC) machine. The optimal parameters influenced the rate of metal removal, the roughness of the machined surface, and the force of cutting. This experimental investigation deals with the optimization of speed (800 rpm, 1200 rpm, and 1600 rpm) and feed (0.15, 0.20, and 0.25 mm/rev) in addition to cutting depth (1.0, 1.5, and 2.0 mm) on the turning of Aluminum 7075 alloy in a CNC machine. The outcome in terms of results such as the removal rate of material (maximum), roughness on the machined surface (minimum), along with cutting force (least amount) were improved by the L27 array Taguchi method. There were 27 specimens of Al7075 alloy produced as per the array, and the corresponding responses were measured with the help of various direct contact and indirect contact sensors. Results were concluded all the way through diagrams of main effects in favor of signal-to-noise ratios and diagrams of surfaces with contour diagrams for various combinations of responses.

SAP97 polymorphisms associated with early onset Parkinson's disease.

OBJECTIVE: To investigate the relationship between SAP97 genetic polymorphisms and sporadic Parkinson's disease (PD) in Han Chinese population with the expectation of offering genetic data for the early prevention and treatment of the disease. METHODS: In this study, we genotyped single-nucleotide polymorphisms (SNPs) (rs3915512 and rs9843659) in theSAP97 gene in 317 patients with PD and 317 healthy-matched controls in a Han Chinese population through the improved multiplex ligation detection reaction (imLDR) technique. Then, we analyzed the association of each SNP, alone or in combination, with risk or age of onset of PD. RESULTS: The SAP97 rs3915512 and rs9843659 polymorphisms were not associated with the risk of PD. However, the minor allele of the rs3915512 and rs9843659 were significantly more common in PD patients with an early age of onset. Additionally, significant differences in the distribution of the onset age of the PD among different genotypes of the rs9843659 polymorphism. The CA haplotype was significantly related to early onset PD. CONCLUSIONS: Our data are the first to suggest that the SAP97 SNPs rs3915512 and rs9843659 and the CA haplotype may be significantly associated with early onset PD in China.

Association of the Synapse-Associated Protein 97 (SAP97) Gene Polymorphism With Neurocognitive Function in Schizophrenic Patients.

The SAP97 gene is located in the schizophrenia susceptibility locus 3q29, and it encodes the synaptic scaffolding protein that interacts with the N-methyl-D-aspartate (NMDA) receptor, which is presumed to be dysregulated in schizophrenia. In this study, we genotyped a single-nucleotide polymorphism (SNP) (rs3915512) in the SAP97 gene in 1114 patients with schizophrenia and 1036 healthy-matched controls in a Han Chinese population through the improved multiplex ligation detection reaction (imLDR) technique. Then, we analyzed the association between this SNP and the patients' clinical symptoms and neurocognitive function. Our results showed that there were no significant differences in the genotype and allele frequencies between the patients and the controls for the rs3915512 polymorphism. However, patients with the rs3915512 polymorphism TT genotype had higher neurocognitive function scores (list learning scores, symbol coding scores, category instances scores and controlled oral word association test scores) than the subjects with the A allele (P = 4.72 × 10-5, 0.027, 0.027, 0.013, respectively). Our data are the first to suggest that the SAP97 rs3915512 polymorphism may affect neurocognitive function in patients with schizophrenia.

Optimization and partial characterization of intracellular anticandidal protein from Aspergillus giganteus MTCC 8408 using taguchi DOE.

A new intracellular antifungal protein (afp) production with average molecular weight 24.3 kDa and yield of 0.65 ± 0.1 mg/gram dry cell weight (gdcw) of mycelia in submerged fermentation of Aspergillus giganteus MTCC 8408 was optimized. Taguchi's DOE (design of experiment) L27 orthogonal array (OA) was constructed using Qualitek-4 software with 8 most influensive factors namely, culture pH, temperature, slant age, inoculum volume, agitation and KH2PO4. Scanning electron microscopy (SEM) was used to correlate the effect of selected factors on fungal cell morphology and afp production. The crude protein purification was accomplished using pure ammonium sulfate fractionation followed by carboxymethyl cellulose (CMC) ion-exchange chromatography and sephadex G-100 gel filtration. The average molecular mass of the purified protein was figured by silver stained SDS (sodium dodecylsulphate)-PAGE (poly-acryl amide gel electrophoresis). In vitro antifungal susceptibility assay was profiled against Candida albicans NCIM 3471 and minimum inhibitory concentrations (MICs) were in the range 3 to 4 µg/ml. Characterization of protein was observed with FTIR (Fourier transform infrared spectroscopy) analysis. The optimal production condition for crude afp was obtained as follows: soluble starch: 20 g/l; Corn steep liquor (CSL, 2%) + proteose peptone (PP, 1%): 30 g/l; pH: 5.8; temperature: 25°C; slant age: 3 d; inoculum size: 5% (v/v); agitation: 180 rpm; KH2PO4: 0.1 g/l. The validation experiments using optimized conditions confirmed an improvement in afp production by 59.4% against the expected enhancement of afp production by 61.22%. The present statistical optimization study revealed an opportunity to promote economical design at the industrial level for future scale up of effective antifungal agent against opportunistic oral and vaginal infection.

Cloning of ribosomal protein L27 cDNA from Jilin Shuangyang sika deer / 中草药

Object To develop the gene resources of Jilin Shuangyang sika deer,a rare animal in China and reveal the pharmacological functions of deer medicinal material on the molecular level. Methods A cDNA plasmid library of spleen cells derived from Shuangyang sika deer was set up. Some housekeeping genes were cloned and analyzed. Results One of the cDNA sequences and it's deduced amino acid sequence in deer share high homology with a group of cDNA sequences encoding ribosomal protein L27 in human,rat,Canis and mouse and their corresponding amino acid sequence respectively. It has the completed open reading frame as well. Conclusion A novel full-length cDNA encoding Shuangyang sika deer ribosomal protein L27 is discovered. This sequence has been deposited in Genbank under accession number AF373231.