The emergence of plasmid-borne cfr-mediated linezolid resistant-staphylococci in Vietnam.

OBJECTIVES: Linezolid is one of the last resort antibiotics effectively used in the treatment of infections caused by multidrug-resistant Gram-positive bacteria. Recent outbreaks of Linezolid resistance have been the great concern worldwide, while many countries have not experienced it. In this work, we aimed to evaluate the existence of linezolid resistance and further clarify potential resistance mechanism(s) in staphylococcal isolates obtained from the hospital in Vietnam, a country in which linezolid resistance had not been previously detected. METHODS: Seventy staphylococcal clinical isolates including MRSA (n=63) and methicillin-resistant coagulase-negative staphylococci (MRCNS, n=7) were collected and analyzed for linezolid resistance. Linezolid-resistant isolates were submitted for whole genome sequencing to search for the resistance determinants. RESULTS: We identified two coagulase-negative staphylococcal isolates that were resistant to linezolid. Whole genome sequencing revealed several alterations in the 23S rRNA and L3, L17, L22, L24, L30 ribosomal proteins. Importantly, both isolates harbour the chloramphenicol/florfenicol resistance (cfr) gene on a plasmid. The plasmid was closely identical to the pLRSA417 plasmid that was originally reported in China. CONCLUSIONS: To the best of our knowledge, this is the first report of cfr-mediated linezolid resistance in clinically isolated staphylococci in Vietnam. We suggest that adequate surveillance is necessary to monitor the dissemination of linezolid resistance among staphylococcal species and other important pathogens.

Requirement of GTP binding for TIF-90-regulated ribosomal RNA synthesis and oncogenic activities in human colon cancer cells.

Transcription initiation factor 90 (TIF-90), an alternatively spliced variant of TIF-IA, differs by a 90 base pair deletion of exon 6. TIF-90 has been shown to regulate ribosomal RNA (rRNA) synthesis by interacting with polymerase I (Pol I) during the initiation of ribosomal DNA (rDNA) transcription in the nucleolus. Recently, we showed that TIF-90-mediated rRNA synthesis can play an important role in driving tumorigenesis in human colon cancer cells. Here we show that TIF-90 binds GTP at threonine 310, and that GTP binding is required for TIF-90-enhanced rRNA synthesis. Overexpression of activated AKT induces TIF-90 T310, but not a GTP-binding site (TIF-90 T310N) mutant, to translocate into the nucleolus and increase rRNA synthesis. Complementing this result, treatment with mycophenolic acid (MPA), an inhibitor of GTP production, dissociates TIF-90 from Pol I and hence abolishes AKT-increased rRNA synthesis by way of TIF-90 activation. Thus, TIF-90 requires bound GTP to fulfill its function as an enhancer of rRNA synthesis. Both TIF variants are highly expressed in colon cancer cells, and depletion of TIF-IA expression in these cells results in significant sensitivity to MPA-inhibited rRNA synthesis and reduced cell proliferation. Finally, a combination of MPA and AZD8055 (an inhibitor of both AKT and mTOR) synergistically inhibits rRNA synthesis, in vivo tumor growth, and other oncogenic activities of primary human colon cancer cells, suggesting a potential avenue for the development of therapeutic treatments by targeting the regulation of rRNA synthesis by TIF proteins.

Ebp1 p48 promotes oncogenic activities in human colon cancer cells through regulation of TIF-90-mediated ribosomal RNA synthesis.

The ErbB3-binding protein 1 (Ebp1) has been reported as either an oncogenic regulator or a tumor suppressor in a variety of cancers. Here, we show that Ebp1 p48, a predominant expression isoform, is highly expressed in the majority of human colon tumor cells compared with normal adjacent tissues and its expression is required for the oncogenic activities of these cells. Depletion of Ebp1 expression in primary colon cancer cells inhibits cell proliferation, colony forming, and invasion in vitro as well as tumor formation in vivo and enhances cell sensitivity to irradiation. We further demonstrated that Ebp1 interacts with TIF-90, a splice variant of transcription initiation factor IA (TIF-IA) of the RNA polymerase I complex, allowing for regulation of ribosomal RNA (rRNA) synthesis and oncogenesis in human colon cancer cells. Moreover, Ebp1 expression is essential for Akt protected TIF-90 stability by preventing TIF-90's ubiquitination by Mdm2 and hence, its proteasomal degradation. The results of the present study support a mechanism of underlying oncogenic activities by means of Ebp1 through regulation of TIF-90-mediated rRNA synthesis and suggest the potential therapeutic treatment of colon cancer by targeting Ebp1 and its signaling.

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML.

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.

The role of ErbB3 binding protein 1 in cancer: Friend or foe?

ErbB3, a member of the epidermal growth factor receptor family, reportedly plays an essential role in the regulation of cancer progression and therapeutic resistance. Numerous studies have indicated that ErbB3 binding protein 1 (Ebp1), a binding partner for ErbB3, plays an important regulatory role in the expression and function of ErbB3, but there is no agreement as to whether Ebp1 also has an ErbB3-independent function in cancer and how it might contribute to tumorigenesis. In this review, we will discuss the different functions of the two Ebp1 isoforms, p48 and p42, that may be responsible for the potentially dual role of Ebp1 in cancer growth.

Trauma and syncope-evidence for further sleep study? A case report.

We report on an 83-year-old male with traumatic brain injury after syncope with a fall in the morning. He had a history of seizures, coronary artery disease and paroxysmal atrial fibrillation (AF). No medical cause for seizures and syncope was determined. During rehabilitation, the patient still complained of seizures, and also reported sleepiness and snoring. Sleep apnea diagnostics revealed obstructive sleep apnea (SA) with an apnea-hypopnoea index of 35/h, and sudden onset of tachycardia with variations of heart rate based on paroxysmal atrial fibrillation. Additional tests showed nocturnal AF which spontaneously converted to sinus rhythm mid-morning with an arrest of 5 s (sick sinus syndrome) and seizures. A DDD-pacer was implanted and no further seizures occurred. SA therapy with nasal continuous positive airway pressure was refused by the patient. Our findings suggests that screening for SA may offer the possibility to reveal causes of syncope and may introduce additional therapeutic options as arrhythmia and SA often occur together which in turn might be responsible for trauma due to syncope episodes.

Generation and characterization of soluble interleukin-33 receptor fused with immunoglobulin gamma-1 constant domain expressed by Pichia pastoris yeast.

OBJECTIVES: Interleukin (IL)-33 is a novel member of pro-inflammatory cytokine IL-1 family, which plays an important role in the immune response. IL-33 was proved to involve in many inflammatory and allergic diseases, thus the inhibition of this cytokine may be a promising treatment for these diseases. Arms of the study were to generate mouse soluble IL-33 receptor fused with human IgG1 Fc domain (msIL33R-Fc) expressed by Pichia pastoris yeast and to characterize the IL-33 inhibitory activity of this protein. METHODS: Clone of P. pastoris expressing msIL33R-Fc was established and the recombinant protein was harvested from culture supernatant by protein A sepharose beads. Recombinant msIL33R-Fc was analysed by SDS-PAGE and Western blotting and activity of the protein was investigated using the immunoprecipitation and the bio-assay on EL-4 cells. KEY FINDINGS: P. pastoris-derived msIL33R-Fc was expressed as a glyco-protein and perhaps in dimeric form. The glycosylation of protein expressed by P. pastoris yeast was more intensive and more heterogeneous compared with the counterpart protein expressed from HEK293 cells. Similar to HEK293-derived protein, msIL33R-Fc from P. pastoris was able to capture IL-33 and to interfere with the interaction between IL-33 and IL-33R in in-vitro condition. In IL-33-stimulated EL-4 cell bio-assay, P. pastoris-derived msIL33R-Fc suppressed IL-33 activity similarly as HEK293-derived msIL33R-Fc did. CONCLUSIONS: P. pastoris yeast can express and secrete bio-functional fusion protein sIL33R-Fc IgG1 and this expression system may be beneficial in future studies on the fusion protein.

Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation.

IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.