Ribosomal Protein L11 Research Articles

The transcribed centromeric gene OsMRPL15 is essential for pollen development in rice.

Centromeres consist of highly repetitive sequences that are challenging to map, clone, and sequence. Active genes exist in centromeric regions, but their biological functions are difficult to explore owing to extreme suppression of recombination in these regions. In this study, we used the CRISPR/Cas9 system to knock out the transcribed gene Mitochondrial Ribosomal Protein L15 (OsMRPL15), located in the centromeric region of rice (Oryza sativa) chromosome 8, resulting in gametophyte sterility. Osmrpl15 pollen was completely sterile, with abnormalities appearing at the tricellular stage including the absence of starch granules and disrupted mitochondrial structure. Loss of OsMRPL15 caused abnormal accumulation of mitoribosomal proteins and large subunit rRNA in pollen mitochondria. Moreover, the biosynthesis of several proteins in mitochondria was defective, and expression of mitochondrial genes was upregulated at the mRNA level. Osmrpl15 pollen contained smaller amounts of intermediates related to starch metabolism than wild-type pollen, while biosynthesis of several amino acids was upregulated, possibly to compensate for defective mitochondrial protein biosynthesis and initiate consumption of carbohydrates necessary for starch biosynthesis. These results provide further insight into how defects in mitoribosome development cause gametophyte male sterility.

RPL11 promotes non-small cell lung cancer cell proliferation by regulating endoplasmic reticulum stress and cell autophagy

BackgroundAbnormal biogenesis and ribosome free function of ribosomal proteins (RPs) is important for tumorgenesis and development. Ribosomal protein L11 (RPL11) is a component of ribosomal 60 S large subunit with different roles in different cancers. Here, we aimed to unravel the role of RPL11 in non-small cell lung cancer (NSCLC), especially those affecting cell proliferation.MethodsRPL11 expression in NCI-H1650, NCI-H1299, A549 and HCC827 and normal lung bronchial epithelial cells HBE was detected using western blotting. The function of RPL11 in NSCLC cells were determined by investigating cell viablity, colony formation and cell migration. Mechanism expoloration of RPL11 effect on NSCLC cells proliferation was explored using flow cytometry, and the effect on autophagy was investigated by the additon of autophagy inhibitor chloroquine (CQ) and endoplasmic reticulum stress (ERS) inhibitor tauroursodeoxycholic acid (TUDCA).ResultsRPL11 was highly expressed in NSCLC cells. Extopic expression of RPL11 promoted NCI-H1299 and A549 cells proliferation, and migration, and promoted the transition from the G1 phase to the S phase of the cell cycle. Small RNA interference of RPL11 (siRNA) suppressed NCI-H1299 and A549 cells proliferation and migration and arrested the cell cycle in G0/G1 phase. Moreover, RPL11 promoted NSCLC cell proliferation by modulating autophagy and ERS. Expression levels of autophagy and ERS markers were induced by RPL11 overexpression and inhibited by siRPL11. CQ partially suppressed RPL11-induced A549 and NCI-H1299 proliferation: CQ addition reduced RPL11-induced cells viability and clone numbers and reversed the cell cycle process. ERS inhibitor (TUDCA) partially reversed RPL11-induced autophagy.ConclusionTaken together, RPL11 has a tumor-promoting role in NSCLC. It promotes the cell proliferation of NSCLC cells by regulating ERS and autophagy.

Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative.

During cotranslational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the endoplasmic reticulum (ER) membrane. Our cryo-electron microscopy structure of ribosome-Sec61 shows binding of an ordered heterotetrameric translocon-associated protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28S ribosomal RNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-α/β/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the cyclotriazadisulfonamide derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147 resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.

Nested Phosphorothioated Hybrid Primer-Mediated Isothermal Amplification for Specific and Dye-Based Subattomolar Nucleic Acid Detection at Low Temperatures.

Developing dye-based isothermal nucleic acid amplification (INAA) at low temperatures such as 37 °C remains a technical challenge. Here, we describe a nested phosphorothioated (PS) hybrid primer-mediated isothermal amplification (NPSA) assay which only utilizes EvaGreen (a DNA-binding dye) to achieve specific and dye-based subattomolar nucleic acid detection at 37 °C. The success of low-temperature NPSA essentially depends on employing Bacillus smithii DNA polymerase, a strand-displacing DNA polymerase with wide range of activation temperature. However, the NPSA's high efficiency entails nested PS-modified hybrid primers and the additives of urea and T4 Gene 32 Protein. To address the inhibition of urea on reverse transcription (RT), one-tube two-stage recombinase-aided RT-NPSA (rRT-NPSA) is established. By targeting human Kirsten rat sarcoma viral (KRAS) oncogene, NPSA (rRT-NPSA) stably detects 0.2 aM of KRAS gene (mRNA) within 90 (60) min. In addition, rRT-NPSA possesses subattomolar sensitivity to detect human ribosomal protein L13 mRNA. The NPSA/rRT-NPSA assays are also validated to obtain consistent results with PCR/RT-PCR methods on qualitatively detecting DNA/mRNA targets extracted from cultured cells and clinical samples. As a dye-based, low-temperature INAA method, NPSA inherently facilitates the development of miniaturized diagnostic biosensors.

5,6-Dihydrotetrazolo[1,5-c]quinazolines: Toxicity prediction, synthesis, antimicrobial activity, molecular docking, and perspectives.

Antimicrobial resistance is a never-ending challenge, which should be considered seriously, especially when using unprescribed "over-the-counter" drugs. The synthesis and investigation of novel biologically active substances is among the directions to overcome this problem. Hence, 18 novel 5,6-dihydrotetrazolo[1,5-c]quinazolines were synthesized, their identity, purity, and structure were elucidated by elemental analysis, IR, LC-MS, 1 Н, and 13 C NMR spectra. According to the computational estimation, 15 substances were found to be of toxicity Class V, two of Class IV, and only one of Class II. The in vitro serial dilution method of antimicrobial screening against Escherichia coli, Staphylococcus aureus, Klebsiella aerogenes, Pseudomonas aeruginosa, and Candida albicans determined b3, c1, c6, and c10 as the "lead-compounds" for further modifications to increase the level of activity. Substance b3 demonstrated antibacterial activity that can be related to the calculated high affinity toward all studied proteins: 50S ribosomal protein L19 (PDB ID: 6WQN), sterol 14-alpha demethylase (PDB ID: 5TZ1), and ras-related protein Rab-9A (PDB ID: 1WMS). The structure-activity and structure-target affinity relationships are discussed. The targets for further investigations and the anatomical therapeutic chemical codes of drug similarity are predicted.

Thiostrepton, a resurging drug inhibiting the stringent response to counteract antibiotic-resistance and expression of virulence determinants in Neisseria gonorrhoeae.

Due to the increased resistance to all available antibiotics and the lack of vaccines, Neisseria gonorrhoeae (the gonococcus) poses an urgent threat. Although the mechanisms of virulence and antibiotic resistance have been largely investigated in this bacterium, very few studies have addressed the stringent response (SR) that in pathogenic bacteria controls the expression of genes involved in host-pathogen interaction and tolerance and persistence toward antibiotics. In this study, the results of the transcriptome analysis of a clinical isolate of N. gonorrhoeae, after induction of the SR by serine hydroxamate, provided us with an accurate list of genes that are transcriptionally modulated during the SR. The list includes genes associated with metabolism, cellular machine functions, host-pathogen interaction, genome plasticity, and antibiotic tolerance and persistence. Moreover, we found that the artificial induction of the SR in N. gonorrhoeae by serine hydroxamate is prevented by thiostrepton, a thiopeptide antibiotic that is known to interact with ribosomal protein L11, thereby inhibiting functions of EF-Tu and EF-G, and binding of pppGpp synthase I (RelA) to ribosome upon entry of uncharged tRNA. We found that N. gonorrhoeae is highly sensitive to thiostrepton under in vitro conditions, and that thiostrepton, in contrast to other antibiotics, does not induce tolerance or persistence. Finally, we observed that thiostrepton attenuated the expression of key genes involved in the host-pathogen interaction. These properties make thiostrepton a good drug candidate for dampening bacterial virulence and preventing antibiotic tolerance and persistence. The ongoing challenge is to increase the bioavailability of thiostrepton through the use of chemistry and nanotechnology.

Mitochondrial Ribosomal Protein L14 Promotes Cell Growth and Invasion by Modulating Reactive Oxygen Species in Thyroid Cancer.

The mitochondrial ribosomal protein L14 (MRPL14) is encoded by a nuclear gene and participates in mitochondrial protein translation. In this study, we aimed to investigate the role of MRPL14 in thyroid cancer. We investigated the association between MRPL14 expression and clinicopathological features using The Cancer Genome Atlas (TCGA) and Chungnam National University Hospital (CNUH) databases. Functional studies of MRPL14, including proliferation, migration, invasion, mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production, were performed in papillary thyroid cancer (PTC) cell lines (B-CPAP and KTC-1). Based on the TCGA dataset, PTC tissues lost mitochondrial integrity and showed dysregulated expression of overall mitoribosomal proteins (MRPs) compared with normal thyroid tissues. Of 78 MRPs, MRPL14 was highly expressed in thyroid cancer tissues. MRPL14 overexpression was significantly associated with advanced tumor stage, extrathyroidal extension, and lymph node metastasis. MRPL14 increased cell proliferation of thyroid cancer and promoted cell migration via epithelial-mesenchymal transition-related proteins. Moreover, MRPL14 knockdown reduced the expression of oxidative phosphorylation complex IV (MTCO1) and increased the accumulation of ROS. Cotreatment with a ROS scavenger restored cell proliferation and migration, which had been reduced by MRPL14 knockdown, implying that ROS functions as a key regulator of the oncogenic effects of MRPL14 in thyroid cancer cells. Our findings indicate that MRPL14 may promote cell growth, migration, and invasion by modulating ROS in thyroid cancer cells.

Babesia microti Variant With Multiple Resistance Mutations Detected in an Immunocompromised Patient Receiving Atovaquone Prophylaxis.

We report Babesia microti genomic sequences with multiple mutations in the atovaquone-target region of cytochrome b, including a newly identified Y272S mutation, plus 1 mutation of undetermined significance in the azithromycin-associated ribosomal protein L4. The parasite was sequenced from an immunocompromised patient on prophylactic atovaquone for Pneumocystis pneumonia before diagnosis of babesiosis.

The molecular mechanisms of apoptosis accompanied with the epigenetic regulation of the NY-ESO-1 antigen in non-small lung cancer cells treated with decitabine (5-aza-CdR)

Dysregulated epigenetic modifications are common in lung cancer but have been reversed using demethylating agent like 5-Aza-CdR. 5-Aza-CdR induces/upregulates the NY-ESO-1 antigen in lung cancer. Therefore, we investigated the molecular mechanisms accompanied with the epigenetic regulation of NY-ESO-1 in 5-Aza-CdR-treated NCI–H1975 cell line. We showed significant induction of the NY-ESO-1 protein (**p < 0.0097) using Cellular ELISA. Bisulfite-sequencing demonstrated 45.6% demethylation efficiency at the NY-ESO-1 gene promoter region and RT-qPCR analysis confirmed the significant induction of NY-ESO-1 at mRNA level (128-fold increase, *p < 0.050). We then investigated the mechanism by which 5-Aza-CdR inhibits cell proliferation in the NCI–H1975 cell line. Upregulation of the death receptors TRAIL (2.04-fold *p < 0.011) and FAS (2.1-fold *p < 0.011) indicate activation of the extrinsic apoptotic pathway. The upregulation of Voltage-dependent anion-selective channel protein 1 (1.9-fold), Major vault protein (1.8-fold), Bax (1.16-fold), and Cytochrome C (1.39-fold) indicate the activation of the intrinsic pathway. We also observed the differential expression of protein Complement C3 (3.3-fold), Destrin (−5.1-fold), Vimentin (−1.7-fold), Peroxiredoxin 4 (−1.6-fold), Fascin (−1.8-fold), Heme oxygenase-2 (−0.67-fold**p < 0.0055), Hsp27 (−0.57-fold**p < 0.004), and Hsp70 (−0.39-fold **p < 0.001), indicating reduced cell growth, cell migration, and metastasis. The upregulation of 40S ribosomal protein S9 (3-fold), 40S ribosomal protein S15 (4.2-fold), 40S ribosomal protein S18 (2.5-fold), and 60S ribosomal protein L22 (4.4-fold) implied the induction of translation machinery. These results reiterate the decisive role of 5-Aza-CdR in lung cancer treatment since it induces the epigenetic regulation of NY-ESO-1 antigen, inhibits cell proliferation, increases apoptosis, and decreases invasiveness.

Isolation of Cytochrome C for Proteomics with Lindqvist-type Polyiodate Modified Metal Organic Framework

Separation and purification of Cytochrome C (Cyt-C) is important for proteomic. High efficient and selective pretreatment method for Cyt-C in real samples are always needed. Herein, polyniobate (K7H[Nb6O19]·13H2O, Nb6O19) is modified on a metal-organic framework MIL-125(Ti) through intermolecular hydrogen bonds and an aqueous-stable composite Nb6O19/MIL-125(Ti) is successfully prepared to pretreat complex protein sample. Protein adsorption studies have shown that Nb6O19/MIL-125(Ti) can promote the selective adsorption of Cyt-C due to the synergistic effect of electrostatic and hydrogen-bond interactions. At pH=10.0 (Britton-Robinson buffer), the adsorption efficiency of 300 μL 100 μg·mL−1 Cyt-C onto 1.0 mg Nb6O19/MIL-125(Ti) can reach 99.5%. The adsorption behavior of Cyt-C fits well with the Langmuir adsorption model, corresponding to a maximum theoretical adsorption capacity of 168.35 mg·g−1. Using 3 mol·L−1 NaCl as the eluent, a high elution efficiency of 92.19% is obtained. In addition, the results of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirm that Nb6O19/MIL-125(Ti) efficiently adsorbed Cyt-C from scrofa heart extraction. LC-MS/MS spectrometry results show that the purification of Cyt-C reduces the abundance from the 12th to the 154th place after Nb6O19/MIL-125(Ti) treatment. Moreover, low abundant proteins, e.g., Superoxide dismutase 1, IF rod domain-containing protein and Ubiquitin-60S ribosomal protein L40 were considerably enriched. These outcomes confirm the practicability of Nb6O19/MIL-125 (Ti) as a Cyt-C extractant has potential application value in scrofa heart proteomics.

Genomic insight into Campylobacter jejuni isolated from commercial turkey flocks in Germany using whole-genome sequencing analysis.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0-26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain-Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (bla OXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3')-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 β-lactam resistance genes (bla OXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Interaction of Camptothecin Anticancer Drugs with Ribosomal Proteins L15 and L11: A Molecular Docking Study.

The antitumor drug topotecan (TPT) is a potent inhibitor of topoisomerase I, triggering DNA breaks lethal for proliferating cancer cells. The mechanism is common to camptothecins SN38 (the active metabolite of irinotecan) and belotecan (BLT). Recently, TPT was shown to bind the ribosomal protein L15, inducing an antitumor immune activation independent of topoisomerase I. We have modeled the interaction of four camptothecins with RPL15 derived from the 80S human ribosome. Two potential drug-binding sites were identified at Ile135 and Phe129. SN38 can form robust RPL15 complexes at both sites, whereas BLT essentially gave stable complexes with site Ile135. The empirical energy of interaction (ΔE) for SN38 binding to RPL15 is similar to that determined for TPT binding to the topoisomerase I-DNA complex. Molecular models with the ribosomal protein L11 sensitive to topoisomerase inhibitors show that SN38 can form a robust complex at a single site (Cys25), much more stable than those with TPT and BLT. The main camptothecin structural elements implicated in the ribosomal protein interaction are the lactone moiety, the aromatic system and the 10-hydroxyl group. The study provides guidance to the design of modulators of ribosomal proteins L11 and L15, both considered anticancer targets.

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Novel maternal autoantibodies in autism spectrum disorder: Implications for screening and diagnosis.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which early recognition is a major challenge. Autoantibodies against fetal brain antigens have been found in the blood of mothers of children with ASD (m-ASD) and can be transferred to the fetus where they can impact neurodevelopment by binding to fetal brain proteins. This study aims to identify novel maternal autoantibodies reactive against human fetal brain antigens, and explore their use as biomarkers for ASD screening and diagnosis. A custom-made human fetal brain cDNA phage display library was constructed, and screened for antibody reactivity in m-ASD samples from the Simons Simplex Collection (SSC) of the Simons Foundation Autism Research Initiative (SFARI). Antibody reactivity against 6 identified antigens was determined in plasma samples of 238 m-ASD and 90 mothers with typically developing children (m-TD). We identified antibodies to 6 novel University Hasselt (UH)-ASD antigens, including three novel m-ASD autoantigens, i.e., ribosomal protein L23 (RPL23), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calmodulin-regulated spectrin-associated protein 3 (CAMSAP3). Antibody reactivity against a panel of four of these targets was found in 16% of m-ASD samples, compared to 4% in m-TD samples (p = 0.0049). Maternal antibodies against 4 UH-ASD antigens could therefore provide a novel tool to support the diagnosis of ASD in a subset of individuals.

Identification of reference genes for RT-qPCR analysis across kiwifruit species under iron deficiency conditions

Selecting appropriate reference genes is crucial for the accuracy of gene expression analysis. Although rootstock species play vital roles in kiwifruit production, the reference genes has not been identified in kiwifruit roots and across kiwifruit species. Additionally, iron (Fe) deficiency has been frequently observed in kiwifruit orchards, but the reference genes is not validated. Here, we assessed ten candidate reference genes: actin (ACT), actin-depolymerizing factor family protein (ADF), ADP-ribosylation factor (ARF), eukaryotic translation initiation factor 5A (eIF5A), ribosomal protein L19 (RPL19), 40S ribosomal protein S17 (RPS17), suppressor of G2 allele of SKP1 (SGT1), translationally-controlled tumor protein (TCTP), translation initiation factor 2-γ (TIF2-γ) and ubiquitin-conjugating enzyme E2 variant (UBE2V), for their suitability as reliable internal controls in roots, stems and leaves of four kiwifruit species (Actinidia chinensis, A. macrosperma, A. polygama, and A. valvata) under direct and bicarbonate-induced Fe deficiency conditions. The expression stability of the candidate genes was evaluated by four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Based on the comprehensive ranking, we recommended TCTP + UBE2V as the best reference genes for roots, UBE2V + ACT for stems, RPL19 + ARF for leaves, and UBE2V + RPL19 for all the tissues. The reliability of the recommended reference genes was verified by six target genes involved in Fe uptake and transport. These results provide a basis for improving the accuracy of gene expression analysis and elucidating the molecular mechanisms of Fe deficiency adaptation in kiwifruit species.

Ribosomal protein L31 (RPL31) inhibits the proliferation and migration of gastric cancer cells

Gastric cancer (GC) is a digestive tract malignant tumor and causes the third cancer-related mortality in the world. Aberrant expression of Ribosomal Protein L31 (RPL31) has been reported in several human cancers. The aim of this study was to explore the role and possible biological functions of RPL31 in GC. We firstly employed immunohistochemistry to examine RPL31 expression in tumor and para-cancerous tissues. By lentiviral transfection, we successfully constructed an RPL31-knockdown GC cell model and performed functional validation to reveal the effects of RPL31 on proliferation, apoptosis, cycle, migration, and tumor growth. Our data indicated that RPL31 was abundantly expressed in GC tissues and cell lines (AGS and MGC-803). In addition, RPL31 expression was positively correlated with the extent of tumor infiltrate of GC patients. Functionally, silencing RPL31 in AGS and MGC-803 cells significantly limited the ability of proliferation and migration, promoted cell apoptosis. Consistently, RPL31-knockdown GC cells inhibited the growth of xenografts in mice. Moreover, preliminary analysis on the downstream regulation mechanism revealed that RPL31 functioned as a tumor promoter through targeting JAK-STAT signaling pathway. In conclusion, inhibition of abnormally high expression of RPL31 in GC may be a potential therapeutic strategy for this disease.

Proteotyping of Campylobacter jejuni by MALDI-TOF MS and Strain Solution Version 2 Software.

Identification of microorganisms by MALDI-TOF MS has become a popular method in the past 20 years. Strain Solution ver. 2 software appended with MALDI-TOF MS enables accurate discrimination of serotypes and strains beyond the genus and species level by creating a theoretical mass-based database. In this study, we constructed a theoretical mass database with the validated biomarkers to proteotype Campylobacter jejuni. Using 10 strains belonging to Campylobacter spp. available from culture collections and 41 Campylobacter jejuni strains isolated from humans and foods, the ribosomal protein subunits L36, L32, S14, L24, L23, L7/L12, and S11 could be selected as the effective biomarkers for the proteotyping of C. jejuni at MALDI-TOF MS. An accurate database of their theoretical mass-based values was constructed by matching these gene DNA sequences and the observed mass peaks. We attempted to automatically classify 41 strains isolated from nature using this database and Strain Solution ver. 2 software, and 38 strains (93%) were correctly classified into the intended group based on the theoretical mass-based values. Thus, the seven biomarkers found in this study and Strain Solution ver. 2 are promising for the proteotyping of C. jejuni by MALDI-TOF MS.

Brucella effectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.

Comparing proteome changes involved in biofilm formation by Streptococcus mutans after exposure to sucrose and starch.

Streptococcus mutans is a main organism of tooth infections including tooth decay and periodontitis. The aim of this study was to assess the influence of sucrose and starch on biofilm formation and proteome profile of S. mutans ATCC 35668 strain. The biofilm formation was assessed by microtiter plating method. Changes in bacterial proteins after exposure to sucrose and starch carbohydrates were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. The biofilm formation of S. mutans was increased to 391.76% in 1% sucrose concentration, 165.76% in 1% starch, and 264.27% in the 0.5% sucrose plus 0.5% starch in comparison to biofilm formation in the media without sugars. The abundance of glutamines, adenylate kinase, and 50S ribosomal protein L29 was increased under exposure to sucrose. Upregulation of lactate utilization protein C, 5-hydroxybenzimidazole synthase BzaA, and 50S ribosomal protein L16 was formed under starch exposure. Ribosome-recycling factor, peptide chain release factor 1, and peptide methionine sulfoxide reductase MsrB were upregulated under exposure to sucrose in combination with starch. The results demonstrated that the carbohydrates increase microbial pathogenicity. In addition, sucrose and starch carbohydrates can induce biofilm formation of S. mutans via various mechanisms such as changes in the expression of special proteins.

Classification of the mitochondrial ribosomal protein-associated molecular subtypes and identified a serological diagnostic biomarker in hepatocellular carcinoma.

The objective of this study was to sort out innovative molecular subtypes associated with mitochondrial ribosomal proteins (MRPs) to predict clinical therapy response and determine the presence of circulating markers in hepatocellular carcinoma (HCC) patients. Using an unsupervised clustering method, we categorized the relative molecular subtypes of MRPs in HCC patients. The prognosis, biological properties, immune checkpoint inhibitor and chemotherapy response of the patients were clarified. A signature and nomogram were developed to evaluate the prognosis. Enzyme-linked immunosorbent assay (ELISA) measured serum mitochondrial ribosomal protein L9 (MRPL9) levels in liver disease patients and normal individuals. Receiver operating characteristic (ROC) curves were conducted to calculate the diagnostic effect. The Cell Counting Kit 8 was carried out to examine cell proliferation, and flow cytometry was used to investigate the cell cycle. Transwell assay was applied to investigate the potential of cell migration and invasion. Western blot detected corresponding changes of biological markers. Participants were classified into two subtypes according to MRPs expression levels, which were characterized by different prognoses, biological features, and marked differences in response to chemotherapy and immune checkpoint inhibitors. Serum MRPL9 was significantly higher in HCC patients than in normal individuals and the benign liver disease group. ROC curve analysis showed that MRPL9 was superior to AFP and Ferritin in differentiating HCC from healthy and benign patients, or alone. Overexpressed MRPL9 could enhance aggressiveness and facilitate the G1/S progression in HCC cells. We constructed novel molecular subtypes based on MRPs expression in HCC patients, which provided valuable strategies for the prediction of prognosis and clinical personalized treatment. MRPL9 might act as a reliable circulating diagnostic biomarker and therapeutic target for HCC patients.