Protein L Research Articles

RPL22L1, a novel candidate oncogene promotes temozolomide resistance by activating STAT3 in glioblastoma

Aggressiveness and drug resistance are major challenges in the clinical treatment of glioblastoma (GBM). Our previously research reported a novel candidate oncogene ribosomal protein L22 like 1 (RPL22L1). The aim of this study was to elucidate the potential role and mechanism of RPL22L1 in progression and temozolomide (TMZ) resistance of GBM. Online database, tissue microarrays and clinical tissue specimens were used to evaluate the expression and clinical implication of RPL22L1 in GBM. We performed cell function assays, orthotopic and subcutaneous xenograft tumor models to evaluate the effects and molecular mechanisms of RPL22L1 on GBM. RPL22L1 expression was significantly upregulated in GBM and associated with poorer prognosis. RPL22L1 overexpression enhanced GBM cell proliferation, migration, invasion, TMZ resistance and tumorigenicity, which could be reduced by RPL22L1 knockdown. Further, we found RPL22L1 promoted mesenchymal phenotype of GBM and the impact of these effects was closely related to EGFR/STAT3 pathway. Importantly, we observed that STAT3 specific inhibitor (Stattic) significantly inhibited the malignant functions of RPL22L1, especially on TMZ resistance. RPL22L1 overexpressed increased combination drug sensitive of Stattic and TMZ both in vitro and in vivo. Moreover, Stattic effectively restored the sensitive of RPL22L1 induced TMZ resistance in vitro and in vivo. Our study identified a novel candidate oncogene RPL22L1 which promoted the GBM malignancy through STAT3 pathway. And we highlighted that Stattic combined with TMZ therapy might be an effective treatment strategy in RPL22L1 high-expressed GBM patients.

Targeting EBV-encoded products: Implications for drug development in EBV-associated diseases.

Epstein-Barr virus, a human gamma-herpesvirus, has a close connection to the pathogenesis of cancers and other diseases, which are a burden for public health worldwide. So far, several drugs or biomolecules have been discovered that can target EBV-encoded products for treatment, such as Silvestrol, affinity toxin, roscovitine, H20, H31, curcumin, thymoquinone, and ribosomal protein L22. These drugs activate or inhibit the function of some biomolecules, affecting subsequent signalling pathways by acting on the products of EBV. These drugs usually target LMP1, LMP2; EBNA1, EBNA2, EBNA3; EBER1, EBER2; Bam-HI A rightward transcript and BHRF1. Additionally, some promising findings in the fields of vaccines, immunological, and cellular therapies have been established. In this review, we mainly summarise the function of drugs mentioned above and unique mechanisms, hoping that we can help giving insight to the design of drugs for the treatment of EBV-associated diseases.

CREB1 Silencing Protects Against Inflammatory Response in Rats with Deep Vein Thrombosis Through Reducing RPL9 Expression and Blocking NF-κB Signaling.

Apoptosis and inflammation of vascular endothelial cells (VECs) are the most important causes of deep vein thrombosis (DVT). cAMP response element binding protein 1 (CREB1) encodes a transcription factor that binds as a homodimer to the cAMP-responsive element and can promote inflammation. CREB1 is found to be upregulated in the plasma of patients with venous thromboembolism. However, the biological functions of CREB1 in DVT remain unknown. We evaluated the effect of CREB1 in a rat model of inferior vena cava (IVA) stenosis-induced DVT. IVC stenosis resulted in stable thrombus, inflammatory response and CREB1 upregulation, whereas CREB1 knockdown inhibited thrombus and inflammation in DVT rats. In vitro analysis showed that CREB1 knockdown inhibited VEC apoptosis. Mechanistically, CREB1 knockdown reduced Ribosomal protein L9 (RPL9) expression and blocked the NF-κB pathway. Therefore, CREB1 may become a potential therapeutic target of DVT prevention.

Multi-omics analysis of MRPL-13 as a tumor-promoting marker from pan-cancer to lung adenocarcinoma.

As a member of the mitochondrial ribosomal protein family, mitochondrial ribosomal protein L13 (MRPL13) is responsible for synthesizing mitochondrial proteins in cells. Several studies have indicated that MRPL13 is associated with the proliferation cycle, migration ability, apoptosis and autophagy of cancer cells. However, a thorough examination of MRPL13 across cancers remains uncertain. Therefore, we tried to clarify the relationship between MRPL13 and pan-cancer, and verified it in lung adenocarcinoma by various methods. Finally, our research is expected to reveal new targets for pan-cancer treatment and improve the prognosis of cancer patients. Using bioinformatics tools, we quantified the differential expression of MRPL13 between cancer tissues and corresponding or noncorresponding normal tissues across cancers. We also analyzed the relationships between MRPL13 expression levels and several factors, including diagnosis, prognosis, mutation, functional signaling pathways, immune infiltration, RNA modification, and the relationship with cuproptosis-related genes. Furthermore, we studied the relationship between the expression level of MRPL13 across cancers and the change in cancer functional status through single-cell data. In addition, quantitative experiments (PCR and Western blot) proved that the expression of MRPL13 was significantly different between LUAD and control samples. Finally, the effect of knocking out MRPL13 on cancer cells was compared by gene silencing experiments. In summary, we used a combination of bioinformatics and experimental applications to study the potential roles of MRPL13 in cancer. After conducting a multidimensional analysis, we found that the application of MRPL13 multigroup analysis can effectively improve the diagnostic efficiency of various cancers and predict the prognosis of cancer. Moreover, MRPL13 in pan-cancer is related to the cancer immune infiltration pattern, methylation level and cuproptosis-related genes. Furthermore, single-cell data analysis showed that the modules of metastasis, EMT, cell cycle, DNA repair, invasion, DNA damage and proliferation were positively correlated with the expression of MRPL13 in LUAD (Lung adenocarcinoma), while the modules of hypoxia and inflammation were negatively correlated. Moreover, through quantitative experiments, we observed higher expression of MRPL13 in cancer tissues at the RNA or protein level. Knockdown of MRPL13 in LUAD led to decreased cancer cell survival, delayed tumor division and migration, reduced invasion, and increased cancer cell apoptosis. Our study demonstrates the potential of using MRPL13 as a molecular biomarker for diagnosing and suggesting the prognosis of certain malignant tumors. Furthermore, our research shows that MRPL13 may be an effective therapeutic target for lung adenocarcinoma.

Screening of genes encoding proteins that interact with Nrf2: Probing a cDNA library from Mytilus coruscus using a yeast two-hybrid system

The Nuclear factor Erythroid 2-related factor 2 (Nrf2) is the most important endogenous antioxidant factor in organisms, and it has been demonstrated that it exerts extensive control over the immune response by interacting with crucial innate immunity components directly or indirectly. Although Nrf2 has been widely confirmed to be involved in stress resistance in mammals and some fish, its contribution to mollusks oxidative stress resistance has not frequently been documented. In this investigation, total RNA was taken from the digestive gland of M. coruscus, and a cDNA library was constructed and screened using the GATEWAY recombination technology. The Nrf2 cDNA sequence of M. coruscus was cloned into the pGBKT7 vector to prepare the bait plasmid. Using yeast two-hybrid system, after auxotrophic medium screening, sequencing, and bioinformatics analysis, 13 binding proteins that interacted with Nrf2 were finally identified. They were QM-like protein, 40S ribosomal protein S4 (RPS4), ribosomal protein S2 (RPS2), ribosomal protein L12 (RPL12), EF1-alpha mRNA for elongation factor 1 alpha (eEF1-alpha), ferritin, alpha-amylase, trypsin, vdg3, period clock protein, cyclophilin A isoform 1 (CYP A), serine protease CFSP2, histone variant H2A.Z (H2A.Z). For a better understanding the physiological function of Nrf2 in animals and as a potential target for future research on protein roles in Nrf2 interactions, it is crucial to clarify these protein interactions.

Haemonchus contortus HcL6 promoted the Th9 immune response in goat PBMCs by activating the STAT6/PU.1/NF-κB pathway

Th9 cells play a crucial role in parasite immunity. The development of Th9 cells is facilitated by several cytokines. Key transcription factors, such as STAT6, STAT5, and PU.1, are known to enhance IL-9 expression during the Th9 immune response. NF-κB-mediated transduction pathways participate in the induction of IL-9. In a previous study, we unveiled a unique ribosomal protein derived from Haemonchus contortus excretory-secretory proteins (HcESPs) that interact with host Th9 cells. In the present study, the effects of the Haemonchus contortus ribosomal protein L6 domain DE-containing protein (HcL6) on IL-9 secretion, Th9 differentiation, and IL-9 transcription were assessed by employing ELISA, flow cytometry, and qPCR methodologies. The observations revealed the transcriptional upregulation of several key genes within the Th9 immune response pathway. Moreover, silencing STAT6, PU.1, and NF-κB was found to attenuate the Th9 immune response. In this study, we unveiled the Th9 immune response-inducing capabilities of HcL6 and elucidated some of its underlying mechanisms. These findings suggest that HcL6 is an immunostimulatory antigen capable of inducing the Th9 immune response. These insights could prove instrumental in identifying potential candidate antigens for the development of immunoprophylactic strategies against H. contortus infections.

High expression of MRPL13 promotes cell cycle progression and proliferation of gastric cancer cells by inhibiting p53 signaling to affect long-term prognosis

To determine the expression of mitochondrial ribosomal protein L13 (MRPL13) in gastric cancer and its impact on long-term prognosis and explore the possible mechanism. We analyzed MRPL13 expression level in gastric cancer and its association with the patients' prognosis based on the public cancer database the data of 100 gastric cancer patients undergoing radical gastrectomy in our hospital from January, 2014 to October, 2017. We further assessed the effects of MRPL13 overexpression and knockdown on proliferation and cell cycle of gastric cancer MGC-803 and SGC-7901 cells in vitro and on subcutaneous xenograft growth in nude mice. Both bioinformatic analysis and the patients' data demonstrated that the expression level of MRPL13 was significantly higher in gastric cancer than in adjacent tissues (P<0.05) and positively correlated with peripheral blood Ki67, CEA and CA19-9 levels (P<0.05). High expression of MRPL13 was an independent risk factor affecting the 5-year survival rate of gastric cancer patients (HR: 3.284; 95% CI: 1.537-7.016). Gene set enrichment analysis suggested that MRPL13 was involved in cell cycle and p53 signaling. In cultured gastric cancer cells, MRPL13 overexpression significantly promoted cell proliferation, G1/S phase transition and the expressions of cyclin D1 and CDK6 (P<0.05), and MRPL13 knockdown produced the opposite effects (P<0.05). MRPL13 overexpression significantly promoted gastric cancer cell xenograft growth (P<0.05), and MRPL13 knockdown obviously inhibited tumor growth in nude mice (P<0.05). In both cultured gastric cancer cells and the xenografts in nude mice, MRPL13 overexpression significantly decreased while MRPL13 knockdown enhanced the expressions of p53 and p21 (P<0.05). MRPL13 is highly expressed in gastric cancer and affects the long- term prognosis of the patients possibly by inhibiting p53 signaling to promote cancer cell proliferation and cell cycle progression.

The P-Site Loop of the Universally Conserved Bacterial Ribosomal Protein L5 Is Required for Maintaining Both Translation Rate and Fidelity.

The bacterial ribosomal 5S rRNA-binding protein L5 is universally conserved (uL5). It contains the so-called P-site loop (PSL), which contacts the P-site tRNA in the ribosome. Certain PSL mutations in yeast are lethal, suggesting that the loop plays an important role in translation. In this work, for the first time, a viable Escherichia coli strain was obtained with the deletion of the major part of the PSL (residues 73-80) of the uL5 protein. The deletion conferred cold sensitivity and drastically reduced the growth rate and overall protein synthesizing capacity of the mutant. Translation rate is decreased in mutant cells as compared to the control. At the same time, the deletion causes increased levels of -1 frameshifting and readthrough of all three stop codons. In general, the results show that the PSL of the uL5 is required for maintaining both the accuracy and rate of protein synthesis in vivo.

Evaluation of reference genes for accurate normalization of qPCR data under biotic stresses in mulberry (Morus indica L.)

Biotic stresses adversely affect the leaf quality as well as the yield of mulberry (Morus spp.) which is used to feed mulberry silkworm (Bombyx mori L.). The different biotic stresses which are of economic importance in mulberry are tukra caused by mealybug insect, root knot disease by nematode, powdery mildew, and root rot by fungi. Reverse transcription-quantitative PCR (RT-qPCR) is a preferred technique to assess the transcript abundance of target genes associated with the diseases using a suitable reference gene as a normalizer. However, no study has been conducted to identify suitable reference genes during different biotic stresses in mulberry. In the present work, six candidate reference genes were quantified through qPCR during different biotic stresses in mulberry and their stability was compared using geNorm, NormFinder and RefFinder algorithms. The analysis of data revealed that actin was the preferred gene during root rot and root knot nematode infection for normalizing gene expression. Ubiquitin gene was the best-scoring gene for mealybug infestation resulting in tukra disease. PP2A (Protein phosphatase 2A) and RPL3 (60S ribosomal protein L3) were highly stable expressing genes during powdery mildew fungi infection. In the combined analysis of all tested biotic stresses, actin gene was the most preferred one followed by ubiquitin and PP2A as reference genes. This study identified the best scoring reference genes in various biotic stresses which will contribute for the precise quantification of target genes by RT-qPCR in mulberry as a tool to unravel the disease resistance mechanism.

Staphylococcus brunensis sp. nov. isolated from human clinical specimens with a staphylococcal cassette chromosome-related genomic island outside of the rlmH gene bearing the ccrDE recombinase gene complex.

Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.

Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

Identification and validation of a fatty acid metabolism gene signature for the promotion of metastasis in liver cancer.

Metastasis is a fatal status for liver cancer, and the identification of an effective prediction model and promising therapeutic target is essential. Given the known relationship between fatty acid (FA) metabolism and the liver, the present study aimed to investigate dysregulation of genes associated with FA metabolism in liver cancer. Bioinformatics analyses were performed on data from patients with hepatocellular carcinoma (HCC) obtained from The Cancer Genome Atlas database using R software packages. Online public tools such as the Human Protein Atlas, Tumor Immune Single-Cell Hub and the University of Alabama at Birmingham Cancer Data Analysis portal were also utilized. Some essential results were further verified using in vitro experiments using HepG2 liver cancer cells. A signature consisting of three genes associated with the progression and prognosis of HCC and FA metabolism was identified. When samples were scored based on the expression of these genes and divided according to the median value, the higher score group showed a worse outcome and repressive immune microenvironment than the lower score group. Downstream pathways such as hypoxia, IL6/JAK/STAT3 and epithelial-mesenchymal transition were found to be significantly activated in the higher score group. As the core factor in the signature, mitochondrial ribosomal protein L35 (MRPL35) was found to be upregulated in HCC and to have certain impacts on the dysregulation of effective immunity. Further investigations and in vitro experiments indicated that MRPL35 facilitates the migration and invasion abilities of liver cancer, and the resistance of HCC to treatment. These findings have important implications regarding the characteristics and mechanisms of metastasis in liver cancer, and provide a promising signature based on FA metabolism-related genes that may be used to predict outcomes and explored as a novel therapeutic target in liver cancer.

Integrated transcriptomic and proteomic analyses of plerocercoid and adult Spirometra mansoni reveal potential important pathways in the development of the medical tapeworm

BackgroundSpirometra mansoni can parasitize animals and humans through food and water, causing parasitic zoonosis. Knowledge of the developmental process of S. mansoni is crucial for effective treatment; thus, it is important to characterize differential and specific proteins and pathways associated with parasite development.MethodsIn this study, we performed a comparative proteomic analysis of the plerocercoid and adult stages using a tandem mass tag-based quantitative proteomic approach. Additionally, integrated transcriptomic and proteomic analyses were conducted to obtain the full protein expression profiles of different life cycle stages of the tapeworm.ResultsApproximately 1166 differentially expressed proteins (DEPs) were identified in adults versus plerocercoids, of which 641 DEPs were upregulated and 525 were downregulated. Gene Ontology (GO), Clusters of Orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that most DEPs related to genetic information processing and metabolism of energy in adults seem to be more activated. In the plerocercoid stage, compared to metabolism, genetic information processing appears more dynamic. Protein-protein interaction (PPI) revealed six key proteins (phosphomannomutase, glutathione transferase, malate dehydrogenase, cytoplasmic, 40S ribosomal protein S15, ribosomal protein L15 and 60S acidic ribosomal protein P2) that may play active roles in the growth and development of S. mansoni. Finally, the combination of transcriptomic and proteomic data suggested that three pathways (ubiquitin-mediated proteolysis, phagosome and spliceosome) and five proteins closely related to these pathways might have a significant influence in S. mansoni.ConclusionsThese findings contribute to increasing the knowledge on the protein expression profiles of S. mansoni and provide new insights into functional studies on the molecular mechanisms of the neglected medical tapeworm.Graphical

Hprt Serves as an Ideal Reference Gene for qRT-PCR Normalization in Rat DRG Neurons.

To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons. The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression. All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA (Rn18s) and β-actin (Actb) exhibited the most significant changes, while ubiquitin C (Ubc), hypoxanthine phosphoribosyl transferase (Hprt), and mitochondrial ribosomal protein L10 (Mrpl10) showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, Actb was found to be the most unstable reference gene, while Hprt was the relatively most stable reference gene. The use of unstable reference genes Actb and ankyrin repeat domain 27 (Ankrd27) as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene Hprt had small inter-assay variation and high stability. Our observations indicate that Hprt is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.

TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6.

Human tripartite motif protein 5α (TRIM5α) is a well-characterized restriction factor for some RNA viruses, including HIV1-5; however, reports are limited for DNA viruses6,7. Here we demonstrate that TRIM5α also restricts orthopoxviruses and, via its SPRY domain, binds to the orthopoxvirus capsid protein L3 to diminish virus replication and activate innate immunity. In response, several orthopoxviruses, including vaccinia, rabbitpox, cowpox, monkeypox, camelpox and variola viruses, deploy countermeasures. First, the protein C6 binds to TRIM5 via the RING domain to induce its proteasome-dependent degradation. Second, cyclophilin A (CypA) is recruited via interaction with the capsid protein L3 to virus factories and virions to antagonize TRIM5α; this interaction is prevented by cyclosporine A (CsA) and the non-immunosuppressive derivatives alisporivir and NIM811. Both the proviral effect of CypA and the antiviral effect of CsA are dependent on TRIM5α. CsA, alisporivir and NIM811 have antiviral activity against orthopoxviruses, and because these drugs target a cellular protein, CypA, the emergence of viral drug resistance is difficult. These results warrant testing of CsA derivatives against orthopoxviruses, including monkeypox and variola.

CUL3 induces mitochondrial dysfunction via MRPL12 ubiquitination in renal tubular epithelial cells.

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease worldwide and the strongest predictor of mortality in patients with diabetes. Despite its significance, the pathological mechanism underlying the onset and progression of DKD remains incompletely understood. In this study, we have shown that mitochondrial ribosomal protein L12 (MRPL12) plays a significant role in DKD by modulating mitochondrial function. We demonstrated that MRPL12 was mainly ubiquitinated at K150 in renal tubular epithelial cells. We have found that Cullin3 (CUL3), an E3 ubiquitin ligase, directly interacts with MRPL12 and induces the K63-linked ubiquitination of MRPL12, resulting in mitochondrial biosynthesis dysfunction. Moreover, under high-glucose (HG) conditions in renal tubular epithelial cells, we observed up-regulation of CUL3 expression, significant increase in CUL3-mediated ubiquitination of MRPL12 and dysregulation of mitochondrial biosynthesis. Notably, CUL3 knockdown stabilised the MRPL12 protein and protected mitochondrial biosynthesis under HG conditions. Our findings provide novel insight into how CUL3 affects mitochondrial biosynthesis in renal tubular epithelial cells through MRPL12 ubiquitination and suggest a potential therapeutic strategy for DKD in the future.

Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection.

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

Plasma exosomes proteome profiling discovers protein markers associated with the therapeutic effect of Chaihu-Longgu-Muli decoction on temporal lobe epilepsy

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) uses Chaihu-Longgu-Muli decoction (CLMD) to alleviate disease, clear away heat, calm the mind, and temper excitation. It has been widely used for the therapy of neuropsychiatric disorders including epilepsy, dementia, anxiety, insomnia, and depression for several centuries in China. Aim of the studyThis study aims to analyze differentially expressed proteins (DEPs) in the plasma exosomes of patients with temporal lobe epilepsy (TLE) and after the Chaihu-Longgu-Muli Decoction (CLMD) therapy and to explore the biomarkers of TLE and the potential targets of CLMD in treating TLE. Materials and methodsThe plasma exosomes of normal people and patients with TLE before the treatment of oxcarbazepine (OXC) and combined treatment of OXC and CLMD (OXC.CLMD) were harvested. The exosomes were separated from plasma through ultracentrifugation and then identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The DEPs were analyzed by proteomics and then subjected to gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The protein level of key genes was detected using Western blot. A lithium chloride-pilocarpine-induced epilepsy rat model was established and treated with OXC alone, OXC. CLMD, and CLMD alone (low dose and high dose). Neuronal injury in the hippocampal dentate gyrus and ribosomal protein L6 (RPL6) expression in the brain tissues were detected using H&E staining, Nissl staining, and Western blot. ResultsThe proteomic analysis showed several DEPs were present among plasma exosomes in the four groups; DEPs were enriched in epilepsy-related function and pathway. Four key proteins were screened, including RPL6, Nucleolin (NCL), Apolipoprotein A1 (APOA1), and Lactate Dehydrogenase A (LDHA). Among them, RPL6, NCL, and LDHA protein levels were downregulated and APOA1 protein level was upregulated in the plasma exosomes of TLE patients. After OXC and OXC. CLMD treatment, the protein level of RPL6, NCL, and LDHA was increased, and the APOA1 protein level was decreased. Moreover, the RPL6 protein level was further elevated after OXC. CLMD treatment than that after OXC treatment. In the TLE rat model, neuronal degeneration and necrosis in the hippocampal dentate gyrus increased and RPL6 expression level decreased. After the treatment with OXC, OXC. CLMD, and CLMD alone, the degeneration and necrosis of neurons decreased, and the RPL6 expression level was increased; RPL6 upregulation was remarkably obvious after CLMD treatment. ConclusionsRPL6, NCL, LDHA and APOA1 are the DEPs in the plasma exosomes of patients with TLE before and after therapy. RPL6 might be a potential biomarker of CLMD in treating TLE.

Characterization of In Vitro Resistance to Linezolid in Mycobacterium abscessus.

Single-step selection of Mycobacterium abscessus mutants resistant to linezolid yielded high-level resistance at a low frequency that was associated with mutations in 23S rRNA or the ribosomal protein L3. Surprisingly, linezolid-resistant rRNA mutations conferred cross-resistance to several unrelated antibiotics. Low-level linezolid-resistant mutants were isolated at a higher frequency and were due to loss-of-function mutations in the transcriptional regulator MAB_4384, the repressor of the drug efflux pump MmpL5-MmpS5. IMPORTANCE The protein synthesis inhibitor linezolid is used for the treatment of lung disease caused by Mycobacterium abscessus. However, many strains of the bacterium show poor susceptibility to the antibiotic. For most clinical isolates, resistance is not due to mutations in the target of the drug, the ribosome. The mechanism responsible for non-target-related, indirect linezolid resistance is unknown. Here, we analyzed the development of linezolid resistance in the M. abscessus reference strain in vitro. We found, as expected, resistance mutations in the ribosome. In addition, we identified mutations in a system that involves a drug pump, suggesting drug efflux as a mechanism of resistance to linezolid. This finding may inform the analysis of clinical resistance to linezolid. Surprisingly, a subset of linezolid-resistant ribosome mutations conferred cross-resistance to several structurally and mechanistically unrelated drugs, uncovering a novel multidrug resistance mechanism.