SPR Analysis of SUMO-Murine Rap1-Interacting Factor 1 C-Terminal Domain Interaction with G4.

One of the advantages of surface plasmon resonance is its sensitivity and real-time analyses performed by this method. These characteristics allow us to further investigate the interactions of challenging proteins like Rap1-interacting factor 1 (Rif1). Rif1 is a crucial protein responsible for regulating different cellular processes including DNA replication, repair, and transcription. Mammalian Rif1 is yet to be fully characterized, partly because it is predicted to be intrinsically disordered for a large portion of its polypeptide. This protein has recently been the target of research as a potential biomarker in many cancers. Therefore, finding its most potent interacting partner is of utmost importance. Previous studies showed Rif1's affinity towards structured DNAs and amongst them, T6G24 was superior. Recent studies have shown mouse Rif1 (muRif1) C-terminal domain's (CTD) role in binding to G-quadruplexes (G4). There were many concerns in investigating the Rif1 and G4 interaction, which can be minimized using SPR. Therefore, for the first time, we have assessed its binding with G4 at nano-molar concentrations with SPR which seems to be crucial for its binding analyses. Our results indicate that muRif1-CTD has a high affinity for this G4 sequence as it shows a very low KD (6 ± 1 nM).

Characterization of Interaction of the MBP-Tagged MuRif1-C-Terminal Domain with G-Quadruplex DNA by SPR.

One of the main players in the cell-specific replication timing pattern is Rap1 interacting factor-1 (Rif1). Rif1 protein consists of N-terminal and C-terminal domains and an intrinsically disordered region in between. It has been suggested that both N- and C-termini of Rif1 are capable of binding to DNA with particularly high affinity to cruciform DNA structures. In the present study, we expressed, solubilized, and purified the maltose-binding protein-tagged murine Rif1 C-terminal domain (MBP-muRif1-CTD). Biological activity of the purified protein was assessed by the electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). Our results show that the MBP-muRif1-CTD binds G-quadruplex (G4) structure with high affinity (KD 19.0 ± 0.8 nM), as was previously suggested. This study is the first step in investigation of the interaction of MBP-Profinity eXact-muRif1-CTD and G4 by SPR.

G-quadruplex binding protein Rif1, a key regulator of replication timing.

DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing and locations of replication. Among them, Rif1 plays a major role to shape the 'replication domains' that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.

Prevalence and Antibiotic Susceptibility Patterns of Extended-Spectrum ß-Lactamase and Metallo-ß-Lactamase-Producing Uropathogenic Escherichia coli Isolates.

Healthcare professionals worldwide have expressed concern over infections by extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL)-producing bacteria. We evaluated the prevalence of ESBL- and MBL-producing Escherichia coli (E. coli) isolated from community-acquired urinary tract infections (UTIs) and their antibiotic-resistance profiles at 3 private laboratories in Tehran, Iran. E. coli isolates were mostly susceptible to meropenem (90.4%) and imipenem (90.0%), followed by amikacin (89.0%) and gentamicin (84.7%). Moreover, we detected that, of the E. coli isolates, 67 (22.3%) were ESBL producers and 21 (7.0%) of E. coli isolates were MBL positive via the imipenem-ethylenediaminetetraacetic acid (EDTA) combined disc test. This report is the first, to our knowledge, on the prevalence of MBL-producing uropathogenic E. coli (UPEC) strains in Iran. The antibiotic resistance of E. coli isolates revealed that 122 (40.7%) were multidrug resistant. The high number of antibiotic-resistant and ß-lactamase-producing UPEC strains necessitates further attention and consideration, particularly MBL-producing strains.

The antibiotic resistance profiles of bacterial strains isolated from patients with hospital-acquired bloodstream and urinary tract infections.

Treatment of nosocomial infections is becoming difficult due to the increasing trend of antibiotics resistance. Current knowledge on antibiotic resistance pattern is essential for appropriate therapy. We aimed to evaluate antibiotic resistance profiles in nosocomial bloodstream and urinary tract pathogens. A total of 129 blood stream and 300 urinary tract positive samples were obtained from patients referring to Besat hospital over a two-year period (2009 and 2010). Antibiotic sensitivity was ascertained using the Kirby-Bauer disk diffusion technique according to CLSI guidelines. Patient's data such as gender and age were recorded. The ratio of gram-negative to gram-positive bacteria in BSIs was 1.6 : 1. The most prevalent BSI pathogen was Coagulase-Negative Staphylococci (CoNS). The highest resistance rate of CoNS was against penicillin (91.1%) followed by ampicillin (75.6%), and the lowest rate was against vancomycin (4.4%). Escherichia coli was the most prevalent pathogen isolated from urinary tract infections (UTIs). Ratio of gram-negative to gram-positive bacteria was 3.2 : 1. The highest resistance rate of E. coli isolates was against nalidixic acid (57.7%). The present study showed that CoNS and E. coli are the most common causative agents of nosocomial BSIs and UTIs, and control of infection needs to be addressed in both antibiotic prescription and general hygiene.