A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

Background

The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals.

Methodology/Principal Findings

We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst ∼8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs.

Conclusions/Significance

In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress.     

hOGG1 Ser326Cys Polymorphism and Risk of Hepatocellular Carcinoma among East Asians: A Meta-Analysis

Background

The hOGG1 gene encodes a DNA glycosylase enzyme responsible for DNA repair. The Ser326Cys polymorphism in this gene may influence its repair ability and thus plays a role in carcinogenesis. Several case-control studies have been conducted on this polymorphism and its relationship with the risk of hepatocellular carcinoma (HCC) among East Asians. However, their results are inconsistent.

Methods

We performed a meta-analysis of published case-control studies assessing the association of the hOGG1 Ser326Cys polymorphism with HCC risk among East Asians. PubMed, EMBASE, SCI, BIOSIS, CNKI and WanFang databases were searched. A random-effect model was used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs). Analyses were conducted for additive, dominant and recessive genetic models.

Results

Eight studies were identified involving 2369 cases and 2442 controls assessing the association of the hOGG1 Ser326Cys polymorphism with HCC risk among East Asians. Applying a dominant genetic model, only in the Chinese population, the Cys allele was significantly associated with increased risk of HCC (OR 1.56, 95% CI 1.12–2.17). However, two studies influenced this finding according to sensitivity analysis. Furthermore, considerable heterogeneity and bias existed among Chinese studies.

Conclusion

There is limited evidence to support that the hOGG1 Ser326Cys polymorphism is associated with HCC risk among East Asians. Well-designed and large-sized studies are required to determine this relationship.     

In Vivo Imaging with Fluorescent Smart Probes to Assess Treatment Strategies for Acute Pancreatitis

Background and Aims

Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis.

Methods

Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent “smart” probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging.

Results

We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio.

Conclusions

We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.     

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.     

The impact of the C-terminal domain on the interaction of human DNA topoisomerase II α and β with DNA

Background

Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction.

Methodology/Principal Findings

We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes.

Conclusions/Significance

The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme''s K_D for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.     

Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

Background

The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPα, EspPβ, EspPγ, and EspPδ), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown.

Methodology/Principal Findings

The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the β-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His¹²⁷, Asp¹⁵⁶ and Ser²⁶³ in Domain 1 form the catalytic triad of EspP.

Conclusions/Significance

Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPβ and EspPδ.     

Dazl Functions in Maintenance of Pluripotency and Genetic and Epigenetic Programs of Differentiation in Mouse Primordial Germ Cells In Vivo and In Vitro

Background

Mammalian germ cells progress through a unique developmental program that encompasses proliferation and migration of the nascent primordial germ cell (PGC) population, reprogramming of nuclear DNA to reset imprinted gene expression, and differentiation of mature gametes. Little is known of the genes that regulate quantitative and qualitative aspects of early mammalian germ cell development both in vivo, and during differentiation of germ cells from mouse embryonic stem cells (mESCs) in vitro.

Methodology and Principal Findings

We used a transgenic mouse system that enabled isolation of small numbers of Oct4ΔPE:GFP-positive germ cells in vivo, and following differentiation from mESCs in vitro, to uncover quantitate and qualitative phenotypes associated with the disruption of a single translational regulator, Dazl. We demonstrate that disruption of Dazl results in a post-migratory, pre-meiotic reduction in PGC number accompanied by aberrant expression of pluripotency genes and failure to erase and re-establish genomic imprints in isolated male and female PGCs, as well as subsequent defect in progression through meiosis. Moreover, the phenotypes observed in vivo were mirrored by those in vitro, with inability of isolated mutant PGCs to establish pluripotent EG (embryonic germ) cell lines and few residual Oct-4-expressing cells remaining after somatic differentiation of mESCs carrying a Dazl null mutation. Finally, we observed that even within undifferentiated mESCs, a nascent germ cell subpopulation exists that was effectively eliminated with ablation of Dazl.

Conclusions and Significance

This report establishes the translational regulator Dazl as a component of pluripotency, genetic, and epigenetic programs at multiple time points of germ cell development in vivo and in vitro, and validates use of the ESC system to model and explore germ cell biology.     

Physical analyses of E. coli heteroduplex recombination products in vivo: on the prevalence of 5' and 3' patches

Background

Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3′-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands.

Methodology/Principle Findings

This paper re-examines heteroduplex-patch-strand polarity using phage λ and the λdv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for λ recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5′ or 3′ DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation.

Conclusions/Significance

These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.     

Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

Background

DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3.

Methodology/Principal Findings

We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer.

Conclusions/Significance

These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.     

Targeting DNA-PKcs and ATM with miR-101 Sensitizes Tumors to Radiation

Background

Radiotherapy kills tumor-cells by inducing DNA double strand breaks (DSBs). However, the efficient repair of tumors frequently prevents successful treatment. Therefore, identifying new practical sensitizers is an essential step towards successful radiotherapy. In this study, we tested the new hypothesis: identifying the miRNAs to target DNA DSB repair genes could be a new way for sensitizing tumors to ionizing radiation.

Principal Findings

Here, we chose two genes: DNA-PKcs (an essential factor for non-homologous end-joining repair) and ATM (an important checkpoint regulator for promoting homologous recombination repair) as the targets to search their regulating miRNAs. By combining the database search and the bench work, we picked out miR-101. We identified that miR-101 could efficiently target DNA-PKcs and ATM via binding to the 3′- UTR of DNA-PKcs or ATM mRNA. Up-regulating miR-101 efficiently reduced the protein levels of DNA-PKcs and ATM in these tumor cells and most importantly, sensitized the tumor cells to radiation in vitro and in vivo.

Conclusions

These data demonstrate for the first time that miRNAs could be used to target DNA repair genes and thus sensitize tumors to radiation. These results provide a new way for improving tumor radiotherapy.     

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

Background

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.

Methodology and Principal Findings

Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.

Conclusion and Significance

Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.     

The Amidase Domain of Lipoamidase Specifically Inactivates Lipoylated Proteins In Vivo

Background

In the 1950s, Reed and coworkers discovered an enzyme activity in Streptococcus faecalis (Enterococcus faecalis) extracts that inactivated the Escherichia. coli and E. faecalis pyruvate dehydrogenase complexes through cleavage of the lipoamide bond. The enzyme that caused this lipoamidase activity remained unidentified until Jiang and Cronan discovered the gene encoding lipoamidase (Lpa) through the screening of an expression library. Subsequent cloning and characterization of the recombinant enzyme revealed that lipoamidase is an 80 kDa protein composed of an amidase domain containing a classic Ser-Ser-Lys catalytic triad and a carboxy-terminal domain of unknown function. Here, we show that the amidase domain can be used as an in vivo probe which specifically inactivates lipoylated enzymes.

Methodology/Principal Findings

We evaluated whether Lpa could function as an inducible probe of α-ketoacid dehydrogenase inactivation using E. coli as a model system. Lpa expression resulted in cleavage of lipoic acid from the three lipoylated proteins expressed in E. coli, but did not result in cleavage of biotin from the sole biotinylated protein, the biotin carboxyl carrier protein. When expressed in lipoylation deficient E. coli, Lpa is not toxic, indicating that Lpa does not interfere with any other critical metabolic pathways. When truncated to the amidase domain, Lpa retained lipoamidase activity without acquiring biotinidase activity, indicating that the carboxy-terminal domain is not essential for substrate recognition or function. Substitution of any of the three catalytic triad amino acids with alanine produced inactive Lpa proteins.

Conclusions/Significance

The enzyme lipoamidase is active against a broad range of lipoylated proteins in vivo, but does not affect the growth of lipoylation deficient E. coli. Lpa can be truncated to 60% of its original size with only a partial loss of activity, resulting in a smaller probe that can be used to study the effects of α-ketoacid dehydrogenase inactivation in vivo.     

Ex Vivo Estimation of Photoacoustic Imaging for Detecting Thyroid Microcalcifications

Background

The aim of this study was to evaluate the diagnostic utility of PAI at detecting thyroid microcalcifications at 700 nm laser wavelengths.

Methods

This study included 36 resected samples in 18 patients. To evaluate the PA manifestation of microcalcifications in PAI, gray level histogram and co-occurrence matrix (COM) texture parameters were extracted from the 3 fixed ROI US and PA images, respectively, per sample. We compared the textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without microcalcifications.

Results

On specimen US, the mean value (2748.4±862.5) of samples with microcalcifications on specimen radiography was higher than that (1961.9±780.2) of those without microcalcifications (P = 0.007). However, there were no significant differences in textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without when applying both the mean value of the three slices of thyroid specimens and the value of the thyroid specimen slice which had the highest value of the mean values in specimen US.

Conclusion

PAI did not show significant PA contrast on thyroid microcalcifications indicating that the experimental setup and protocols should be enhanced, e.g., method of complete blood rejection from ex vivo specimens, the multi-wavelength spectroscopic PA imaging method which can solely extract the PA signal from microcalcifications even with high spectral interferences, or PA imaging with narrower slice thickness using 2-dimensional array transducer, etc.