Phosphorylation of threonine 190 is essential for nuclear localization and endocytosis of the FTS (Fused Toes Homolog) protein

Fused Toes Homolog (FTS) is a member of a group of proteins termed as E2 variants and this group of proteins lacks an active cysteine residue that is required for ubiquitin transfer. We have identified the expression of this protein in early neoplastic stages of cervical cancer and its translocation into nucleus from cytoplasm upon irradiation. Here we have reported that a threonine residue at position 190 is essential for its nucleocytoplasmic shuttling and function. Upon LMB treatment we found that FTS was located in the nucleus and it suggests that direct role of nuclear export signal (NES) is required for the binding to CRM1 and facilitates nuclear export. The threonine residue was phosphorylated and promoted the phosphorylation of EGFR, p38 and JNK facilitating vesicular trafficking of early to late endosomes. Mutational change of the threonine into alanine resulted in the cytoplasmic localization of FTS and failed to phosphorylate EGFR and its downstream effector proteins. In addition the mutation also reduced the number of early endosomes formed and also resulted in the clustering of late endosomes around the perinuclear region. These data suggest that threonine residue of FTS at position 190 is not only essential for its function but also for the formation, maturation and trafficking of early endosomes to late endosome/lysosome, as well as we speculate that FTS may function at a connection point in the vesicle tethering.     

Fused Toes Homolog modulates radiation cytotoxicity in uterine cervical cancer cells

Radiotherapy is the major treatment modality for uterine cervical cancer, but in some cases, the disease is radioresistant. Defining the molecular events that contribute to radioresistance and progression of cancer are of critical importance. Here we evaluated the role of Fused Toes Homolog (FTS) in radiation resistance of cervical carcinoma. Immunostaning of cervical cancer cells and tissues revealed that FTS localization and expression was changed after radiation. Targeted stable knockdown of FTS in HeLa cells led to the growth inhibition after radiation. Radiation induced AKT mediated cytoprotective effect was countered by FTS knockdown which leads to PARP cleavage and caspase-3 activation leading to cell death. FTS knockdown promotes radiation induced cell cycle arrest at G0/G1 and apoptosis of HeLa cells with concurrent alterations in the display of cell cycle regulatory proteins. This study revealed FTS is involved in radioresistance of cervical cancer. Targeted inhibition of FTS led to the shutdown of key elemental characteristics of cervical cancer and could lead to an effective therapeutic strategy.     

Protective Role of Quercetin on PCBs-Induced Oxidative Stress and Apoptosis in Hippocampus of Adult Rats

Polychlorinated biphenyls (PCBs) exposure produces neurodegeneration and induces oxidative stress. Neuroprotective role of quercetin, on PCBs induced apoptosis in hippocampus has not yet been studied. The present study is focused to see whether quercetin supplementation precludes against PCBs induced oxidative stress and hippocampal apoptosis. The results have shown that quercetin at 50 mg/kg bwt/30 days has protected oxidative stress in hippocampus of adult male rats. Quercetin, a free radical scavenger decreased the levels of oxidative stress markers in the hippocampus of simultaneous PCB+quercetin treated rats. The pro-apoptotic and anti-apoptotic molecules such as Bad, Bid, Bax and Bcl2 were altered in the hippocampus of experimental animals. PCBs increased the DNA damage and induced neurodegeneration were assessed by histological studies. PCB induced ROS may be linked to increased hippocampal neuronal apoptosis. Quercetin supplementation decreased the neuronal damage and scavenged the free radicals induced by PCBs and protects PCBs induced apoptosis and oxidative stress.     

Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A

Single-stranded-DNA-binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence-nonspecific binding of ssDNA, NMR chemical shift and (15)N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone (13)C, (15)N, and (1)H resonances of RPA70A were assigned for the free protein and the d-CTTCA complex. The binding-induced changes in backbone chemical shifts were used to map out the ssDNA-binding site. Comparison to results obtained for the complex with d-C(5) showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R(1) and R(2)) and (1)H-(15)N NOE values were measured for RPA70A in the absence and presence of d-CTTCA. Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence-nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.     

CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog

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Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

In vivo efficacy of tobramycin-loaded synthetic calcium phosphate beads in a rabbit model of staphylococcal osteomyelitis

Background

Osteomyelitis is an acute or chronic inflammatory process of the bone following infection with pyogenic organisms like Staphylococcus aureus. Tobramycin (TOB) is a promising aminoglycoside antibiotic used to treat various bacterial infections, including S. aureus. The aim of this study was to investigate the efficacy of tobramycin-loaded calcium phosphate beads (CPB) in a rabbit osteomyelitis model.

Methods

Tobramycin (30 mg/mL) was incorporated into CPB by dipping method and the efficacy of TOB-loaded CPB was studied in a rabbit osteomyelitis model. For juxtaposition, CPB with and without TOB were prepared. Twenty-five New Zealand white rabbits were grouped (n?=?5) as sham (group 1), TOB-loaded CPB without S. aureus (group 2), S. aureus only (group 3), S. aureus?+?CPB (group 4), and S. aureus?+?TOB-loaded CPB (group 5). Groups infected with S. aureus followed by CPB implantation were immediately subjected to surgery at the mid-shaft of the tibia. After 28 days post-surgery, all rabbits were euthanized and the presence or absence of chronic osteomyelitis and the extent of architectural destruction of the bone were assessed by radiology, bacteriology and histological studies.

Results

Tobramycin-loaded CPB group potentially inhibited the growth of S. aureus causing 3.2 to 3.4 log₁₀ reductions in CFU/g of bone tissue compared to the controls. Untreated groups infected with S. aureus showed signs of chronic osteomyelitis with abundant bacterial growth and alterations in bone architecture. The sham group and TOB-loaded CPB group showed no evidence of bacterial growth.

Conclusions

TOB-incorporated into CPB for local bone administration was proven to be more successful in increasing the efficacy of TOB in this rabbit osteomyelitis model and hence could represent a good alternative to other formulations used in the treatment of osteomyelitis.

     

Acetonitrile-induced conformational transitions in poly-l-lysine

The effect of acetonitrile on the random coil, α-helix and β-sheet conformations induced in poly-

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-lysine is studied. It is found that acetonitrile at higher concentrations transforms the backbone of polylysine from a random coil to a helical conformation. Addition of acetonitrile to polylysine (pH 11.5) in the α-helix conformation, induces conformational changes in two stages. At concentrations below 60% v/v, acetonitrile stabilizes the helical conformation and at higher concentrations (>70% v/v), it destabilizes the helix. β-sheet→α-helix→random coil conformational transitions are found to occur when polylysine in the heat-induced conformation is titrated with acetonitrile. The possible mechanism(s) of action of acetonitrile in inducing these structural transitions is discussed.