Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.

OBJECTIVES: Pseudomonas aeruginosa colonizes the cystic fibrosis (CF) airways causing chronic bacterial lung infections. CF patients are routinely treated with macrolides, however, P. aeruginosa is considered insusceptible as consequence of inadequate susceptibility testing leaving resistance mechanism completely overlooked. Here, we investigated a new mechanism of macrolide resistance caused by ribosomal protein mutations. METHODS: Investigating a longitudinal collection of 529 isolates from CF patients and analysing 5758 protein sequences from different sources, mutations in P. aeruginosa's ribosomal proteins connected to macrolide resistance were identified. Using a modified susceptibility testing protocol, isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics and macrolide-induced quorum sensing modulation. Proteome and ribosome profiling were applied to assess the impact of the mutations on the bacterial physiology. RESULTS: Five uL4 mutations were identified in isolates from different CF patients. Most mapped to the conserved loop region of uL4 and resulted in increased macrolide tolerance (>10-fold relative to wt strains). Greater concentrations (>10-fold) of macrolide antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity of the uL4 mutants. 16 proteins involved in ribosome adaptation displayed altered expression possibly to compensate for the uL4 mutations, which changed the ribosome stoichiometry without negatively affecting bacterial physiology. CONCLUSIONS: Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated when patients are treated with prolonged courses of macrolides. Importantly, improved macrolide susceptibility testing is necessary for the detection of resistant bacteria.

Ribosomal proteins induce stem cell-like characteristics in glioma cells as an "extra-ribosomal function".

The characteristic features of plasticity and heterogeneity in glioblastoma (GB) cells cause therapeutic difficulties. GB cells are exposed to various stimuli from the tumor microenvironment and acquire the potential to resist chemoradiotherapy. To investigate how GB cells acquire stem cell-like phenotypes, we focused on ribosomal proteins, because ribosome incorporation has been reported to induce stem cell-like phenotypes in somatic cells. Furthermore, dysregulation of ribosome biogenesis has been reported in several types of cancer. We focused on ribosomal protein S6, which promotes sphere-forming ability and stem cell marker expression in GB cells. We expect that investigation of dysregulation of ribosome biogenesis and extra-ribosomal function in GB will provide new insights about the plasticity, heterogeneity, and therapeutic resistance of GB cells, which can potentially lead to revolutionary therapeutic strategies.

Ribosomal proteins and human diseases: molecular mechanisms and targeted therapy.

Ribosome biogenesis and protein synthesis are fundamental rate-limiting steps for cell growth and proliferation. The ribosomal proteins (RPs), comprising the structural parts of the ribosome, are essential for ribosome assembly and function. In addition to their canonical ribosomal functions, multiple RPs have extra-ribosomal functions including activation of p53-dependent or p53-independent pathways in response to stress, resulting in cell cycle arrest and apoptosis. Defects in ribosome biogenesis, translation, and the functions of individual RPs, including mutations in RPs have been linked to a diverse range of human congenital disorders termed ribosomopathies. Ribosomopathies are characterized by tissue-specific phenotypic abnormalities and higher cancer risk later in life. Recent discoveries of somatic mutations in RPs in multiple tumor types reinforce the connections between ribosomal defects and cancer. In this article, we review the most recent advances in understanding the molecular consequences of RP mutations and ribosomal defects in ribosomopathies and cancer. We particularly discuss the molecular basis of the transition from hypo- to hyper-proliferation in ribosomopathies with elevated cancer risk, a paradox termed "Dameshek's riddle." Furthermore, we review the current treatments for ribosomopathies and prospective therapies targeting ribosomal defects. We also highlight recent advances in ribosome stress-based cancer therapeutics. Importantly, insights into the mechanisms of resistance to therapies targeting ribosome biogenesis bring new perspectives into the molecular basis of cancer susceptibility in ribosomopathies and new clinical implications for cancer therapy.

Ribosomal proteins as a possible tool for blocking SARS-COV 2 virus replication for a potential prospective treatment.

Coronavirus disease (COVID-19) is caused by SARS-COV2 and has resulted in more than four million cases globally and the death cases exceeded 300,000. Normally, a range of surviving and propagating host factors must be employed for the completion of the infectious process including RPs. Viral protein biosynthesis involves the interaction of numerous RPs with viral mRNA, proteins which are necessary for viruses replication regulation and infection inside the host cells. Most of these interactions are crucial for virus activation and accumulation. However, only small percentage of these proteins is specifically responsible for host cells protection by triggering the immune pathway against virus. This research proposes RPs extracted from bacillus sp. and yeast as new forum for the advancement of antiviral therapy. Hitherto, antiviral therapy with RPs-involving viral infection has not been widely investigated as critical targets. Also, exploring antiviral strategy based on RPs could be a promising guide for more potential therapeutics.

Toward RNA Repair of Diamond Blackfan Anemia Hematopoietic Stem Cells.

Diamond blackfan anemia (DBA) is a well-known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, GATA1, or TSR2, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the RPS19 gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some RPS19 missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem-cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells all exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. The authors have been interested in the application of spliceosome-mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, SMaRT has several potential advantages. First, delivery of the entire normal cDNA is unnecessary, thus minimizing the overall size of the construct for packaging into a viral delivery vector. Second, RNA transcription of the corrected gene relies on the cell's endogenous transcriptional, processing, and regulatory machinery, thereby ensuring faithful and contextual expression. Third, RNA trans-splicing employs the endogenous spliceosome enzymatic machinery present in nearly all cells. Fourth, RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus may be capable of treating disorders caused by dominant negative mutations. This review critically assesses prospects for both gene and RNA repair in DBA stem cells.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 µM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent.

A helix-PXXP-helix peptide with antibacterial activity without cytotoxicity against MDRPA-infected mice.

In response to the growing problem of multidrug-resistant pathogenic microbes, much attention is being paid to naturally occurring and synthetic antimicrobial peptides (AMPs) and the effects of their structural modification. Among these modifications, amino acid substitution is a simple approach to enhancing biological activity and reducing cytotoxicity. An earlier study indicated that HPA3, an analog of HP (2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1, forms large pores and shows considerable cytotoxicity. However, HPA3P, in which a proline (Pro) is substituted for glutamic acid (Glu) at position 9 of HPA3, shows markedly less cytotoxicity. This may be attributable to the presence of a Pro-kink into middle of the HPA3P structure within the membrane environment. Unfortunately, HPA3P is not an effective antibacterial agent in vivo. We therefore designed a helix-PXXP-helix structure (HPA3P2), in which Pro was substituted for the Glu and phenylalanine (Phe) at positions 9 and 12 of HPA3, yielding a molecule with a flexible central hinge. As compared to HPA3P, HPA3P3 exhibited dramatically increased antibacterial activity in vivo. ICR mice infected with clinically isolated multidrug-resistant Pseudomonas aeruginosa showed 100% survival when administered one 0.5-mg/kg dose of HPA3P2 or three 0.1-mg/kg doses of HPA3P2. Moreover, in a mouse model of septic shock induced by P. aeruginosa LPS, HPA3P2 reduced production of pro-inflammatory mediators and correspondingly reduced lung (alveolar) and liver tissue damage. The changes in HPA3 behavior with the introduction of Pro likely reflects alterations of the mechanism of action: i) HPA3 forms pores in the bacterial cell membranes, ii) HPA3P permeates the cell membranes and binds to intracellular RNA and DNA, and iii) HPA3P2 acts on the outer cellular membrane component LPS. Collectively, these results suggest HPA3P2 has the potential to be an effective antibiotic for use against multidrug-resistant bacterial strains.

Overexpression, purification, molecular characterization and pharmacological evaluation for anticancer activity of ribosomal protein S23 from the giant panda (Ailuropoda melanoleuca).

Ribosomal protein S23 (RPS23) is a component of the 40S small ribosomal subunit encoded by the RPS23 gene, which is specific to eukaryotes. The cDNA and genomic sequence of RPS23 were cloned from Ailuropoda melanoleuca (A. melanoleuca) using reverse transcription­polymerase chain reaction (RT-PCR) technology and touchdown PCR, respectively. The two sequences were analyzed preliminarily and the cDNA of the RPS23 gene was overexpressed in Escherichia coli (E. coli) BL21. The cDNA of RPS23 cloned from giant panda was 472 bp, and it contained an open reading frame (ORF) of 432 bp encoding 142 amino acids. The nucleotide sequence of the coding sequence showed a high degree of homology to some mammals as determined by BLAST analysis, similar to the amino acid sequence. The genomic sequence was 2,105 bp in length, with 4 exons and 3 introns. The primary structure analysis revealed that the molecular weight of the putative RPS23 protein was 15.80 kDa with a theoretical isoelectric point (pI) of 11.23. The molecular weight of the recombinant protein RPS23 was 21.5 kDa with a theoretical pI of 10.57. Topology prediction showed that there are seven different patterns of functional sites in the RPS23 protein of giant panda. RPS23 was successfully expressed in E. coli and its protein fused with the N­terminal His­tagged protein triggered the accumulation of an expected 21.5­kDa polypeptide. The inhibitory rate of tumor growth in mice treated with 0.1 µg/ml RPS23 protein was 49.45%, the highest in the three doses used, which may be comparable to mannatide treatment. Histology of immune organs showed that the tissues were characterized by a regular and tight arrangement, while tumor tissues of the mice in the RPS23 group exhibited a loose arrangement compared to the control group. However, there was no obvious damage to other organs, such as the heart, lung and kidney. Investigations are currently being conducted to determine the bioactive principles of the recombinant protein RPS23 responsible for its anticancer activity.

Ribosomal protein S19 is a novel therapeutic agent in inflammatory kidney disease.

RPS19 (ribosomal protein S19), a component of the 40S small ribosomal subunit, has recently been identified to bind the pro-inflammatory cytokine macrophage MIF (migration inhibitory factor). In vitro experiments identify RPS19 as the first endogenous MIF inhibitor by blocking the binding of MIF to its receptor CD74 and MIF functions on monocyte adherence to endothelial cells. In the present study, we sought to establish whether recombinant RPS19 can exert anti-inflammatory effects in a mouse model of anti-GBM (glomerular basement membrane) GN (glomerulonephritis) in which MIF is known to play an important role. Accelerated anti-GBM GN was induced in C57BL/6J mice by immunization with sheep IgG followed 5 days later by administration of sheep anti-mouse GBM serum. Groups of eight mice were treated once daily by intraperitoneal injection with 6 mg of RPS19/kg of body weight or an irrelevant control protein (human secretoglobin 2A1), or received no treatment, from day 0 until being killed on day 10. Mice that received control or no treatment developed severe crescentic anti-GBM disease on day 10 with increased serum creatinine, declined creatinine clearance and increased proteinuria. These changes were associated with up-regulation of MIF and its receptor CD74 activation of ERK (extracellular-signal-regulated kinase) and NF-κB (nuclear factor κB) signalling, prominent macrophage and T-cell infiltration, as well as up-regulation of Th1 [T-bet and IFNγ (interferon γ)] and Th17 [STAT3 (signal transducer and activator of transcription 3) and IL (interleukin)-17A] as well as IL-1ß and TNFα (tumour necrosis factor α). In contrast, RPS19 treatment largely prevented the development of glomerular crescents and glomerular necrosis, and prevented renal dysfunction and proteinuria (all P<0.001). Of note, RPS19 blocked up-regulation of MIF and CD74 and inactivated ERK and NF-κB signalling, thereby inhibiting macrophage and T-cell infiltration, Th1 and Th17 responses and up-regulation of pro-inflammatory cytokines (all P<0.01). These results demonstrate that RPS19 is a potent anti-inflammatory agent, which appears to work primarily by inhibiting MIF signalling.

Transduced PEP-1-ribosomal protein S3 (rpS3) ameliorates 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice.

This study investigated the preventive effect of ribosomal protein S3 (rpS3) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. A cell permeable expression vector PEP-1-rpS3 was constructed. Topical application of the vector markedly inhibited TPA-induced expression levels of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines. Application of PEP-1-rpS3 also resulted in a significant reduction in the activation of nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) in TPA-treated ears. These results indicate that PEP-1-rpS3 inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK, prompting the suggestion that PEP-1-rpS3 can be used as a therapeutic agent against skin inflammation.

How nature morphs peptide scaffolds into antibiotics.

The conventional notion that peptides are poor candidates for orally available drugs because of protease-sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide-based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best-selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic-resistant bacterial pathogens.

[Imaging of inflammatory foci with 99m technecium labeled antimicrobial oligopeptide].

A direct method using 99mtechnecium to label the synthetic antimicrobial oligopeptide ubiquicidine with a molecular mass of 1.7 kDa has been developed. The radiochemical purity of the resultant labeled compound was more than 98%. A combination of ubiquicidine and 99mtechnecium showed stability in blood plasma. The biological study indicated that the complex was renally excreted from blood flow rapidly (the half-life is 30 min). It accumulated in the experimental inflammatory focus of laboratory animals, peaking 1-2 hours after intravenous injection when the inflammatory focus/intact tissue ratio amounted up to 2.6-3.4, which could yield satisfactory abscess scintigrams. The findings make it possible to consider ubiquicidine as a promising compound for the design of radiopharmaceutical to image inflammatory foci.

Targeted diagnosis and treatment of superficial bladder cancer with monoclonal antibody BDI-1.

OBJECTIVE: To explore the application of monoclonal antibody (McAb) to targeted treatment of bladder carcinoma through a series of in vitro and in vivo studies carried out in animal model and patients with bladder carcinoma. METHODS: Monoclonal antibody BDI-1 against bladder carcinoma was prepared by the lymphocyte hybridoma technique. McAb was conjugated with 99mTc by direct reduction method. Momodin (MD) was covalently linked to McAb by SPDP method. Radioimmunoimaging of nude mice xenografts and patients with bladder carcinoma were performed with BDI-1-99mTc conjugates. An immunotoxin (BDI-1-MD) was inducted via a catheter into the bladder. Targeted treatment with BDI-1-MD was carried out in 18 patients. RESULTS: This study showed the specificity of McAb, and clear imaging of nude mice bearing xenografts. Distribution analysis of 99mTc-BDI-1 in nude mice showed the highest value of T/NT in bladder tumor. Targeted diagnosis and treatment for patients by intravesical administration are very safe and effective. CONCLUSION: The bladder cancer seems an ideal model for diagnostic and therapeutic approaches using regional administration of McAb conjugates via a catheter direct into the bladder.

Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice.

The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.

[The isolation of the ribosomal fraction of Clostridium perfringens type A and an evaluation of its protective activity]. / Poluchenie ribosomal'noi fraktsii Clostridium perfringens tipa A i otsenka ee protektivnoi aktivnosti.

A method for isolation of the ribosomal fraction (RF) from the cytoplasm of type-A C. perfringens strain BP6K was developed and its chemical and antigenic properties characterized. RF has been found to possess protective properties: two subcutaneous immunizations of mice with RF preparations adsorbed on Al(OH)3 in doses of 250 and 500 micrograms (dry weight) has ensured, on the average, the protection of 41.9% of the immunized animals from 1 DCL of type-A C. perfringens strain BP6K culture.

[Thymectomy and Ribomunyl: apropos of 2 cases]. / Thymectomie et Ribomunyl: à propos de 2 observations.

Two cases of post-thymectomy acquired immunodeficiency are presented, together with its clinical and laboratory control, after stimulation with bacterial ribosomal vaccines (Ribomunyl).