RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.     

Factors Influencing the Degradation of Archival Formalin-Fixed Paraffin-Embedded Tissue Sections

The loss of antigenicity in archival formalin-fixed paraffin-embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Sampling-rate-dependent probabilistic Boolean networks

External control of a genetic regulatory network is used for the purpose of avoiding undesirable states, such as those associated with a disease. To date, intervention has mainly focused on the external control of probabilistic Boolean networks via the associated discrete-time discrete-space Markov processes. Implementation of an intervention policy derived for probabilistic Boolean networks requires nearly continuous observation of the underlying biological system since precise application requires the observation of all transitions. In medical applications, as in many engineering problems, the process is sampled at discrete time intervals and a decision to intervene or not must be made at each sample point. In this work, sampling-rate-dependent probabilistic Boolean network is proposed as an extension of probabilistic Boolean network. The proposed framework is capable of capturing the sampling rate of the underlying system.     

Evidence for significant heritability of apoptotic and cell cycle responses to ionising radiation

Genetic factors are likely to affect individual cancer risk, but few quantitative estimates of heritability are available. Public health radiation protection policies do not in general take this potentially important source of variation in risk into account. Two surrogate cellular assays that relate to cancer susceptibility have been developed to gain an insight into the role of genetics in determining individual variation in radiosensitivity. These flow cytometric assays for apoptosis induction and cell cycle delay following radiation are sufficiently sensitive to distinguish lymphocytes from a healthy donor population from those of a sample of obligate carriers of ATM mutations (P = 0.01 and P = 0.02, respectively). Analysis of 54 unselected twin pairs (38 dizygotic, 16 monozygotic) indicated much greater intrapair correlation in response in monozygotic than in dizygotic pairs. Structural equation modelling indicated that models including unique environmental factors only fitted the data less well than those incorporating two or more of additive genetic factors, common environmental factors and unique environmental factors. A model incorporating additive genetic factors and unique environmental factors yielded estimates of heritability for the two traits of 68% (95% CI 40–82%, cell cycle) and 59% (95% CI 22–79%, apoptosis). Thus, these data suggest that genetic factors contribute significantly to human variation in these two measures of radiosensitivity that relate to cancer susceptibility.     

A new two-color Fab labeling method for autoantigen protein microarrays

Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.     

Saccade reward signals in posterior cingulate cortex

Movement selection depends on the outcome of prior behavior. Posterior cingulate cortex (CGp) is strongly connected with both limbic and oculomotor circuitry, and CGp neurons respond following saccades, suggesting a role in signaling the motivational outcome of gaze shifts. To test this hypothesis, single CGp neurons were studied in monkeys while they shifted gaze to visual targets for liquid rewards that varied in size or were delivered probabilistically. CGp neurons responded following saccades as well as following reward delivery, and these responses were correlated with reward size. CGp neurons also responded following the omission of predicted rewards. The timing of CGp activation and its modulation by reward could provide signals useful for updating representations of expected saccade value.     

Mature peripheral RPE cells have an intrinsic capacity to proliferate; a potential regulatory mechanism for age-related cell loss

Background

Mammalian peripheral retinal pigmented epithelium (RPE) cells proliferate throughout life, while central cells are senescent. It is thought that some peripheral cells migrate centrally to correct age-related central RPE loss.

Methodology/Principal Findings

We ask whether this proliferative capacity is intrinsic to such cells and whether cells located centrally produce diffusible signals imposing senescence upon the former once migrated. We also ask whether there are regional differences in expression patterns of key genes involved in these features between the centre and the periphery in vivo and in vitro. Low density RPE cultures obtained from adult mice revealed significantly greater levels of proliferation when derived from peripheral compared to central tissue, but this significance declined with increasing culture density. Further, exposure to centrally conditioned media had no influence on proliferation in peripheral RPE cell cultures at the concentrations examined. Central cells expressed significantly higher levels of E-Cadherin revealing a tighter cell adhesion than in the peripheral regions. Fluorescence-labelled staining for E-Cadherin, F-actin and ZO-1 in vivo revealed different patterns with significantly increased expression on central RPE cells than those in the periphery or differences in junctional morphology. A range of other genes were investigated both in vivo and in vitro associated with RPE proliferation in order to identify gene expression differences between the centre and the periphery. Specifically, the cell cycle inhibitor p27^Kip1 was significantly elevated in central senescent regions in vivo and mTOR, associated with RPE cell senescence, was significantly elevated in the centre in comparison to the periphery.

Conclusions

These data show that the proliferative capacity of peripheral RPE cells is intrinsic and cell-autonomous in adult mice. These differences between centre and periphery are reflected in distinct patterns in junctional markers. The regional proliferation differences may be inversely dependent to cell-cell contact.     

Inhibition of semaphorin 4D enhances chemosensitivity by increasing 5-fluorouracile-induced apoptosis in colorectal cancer cells

Molecular Biology Reports - Overexpression of semaphorin 4D (SEMA4D), an immune semaphorin, is found in various human malignancies, including colorectal cancer (CRC). In this study, we explored the...     

Activity-induced Notch signaling in neurons requires Arc/Arg3.1 and is essential for synaptic plasticity in hippocampal networks

Notch signaling in the nervous system has been most studied in the context of cell fate specification. However, numerous studies have suggested that Notch also regulates neuronal morphology, synaptic plasticity, learning, and memory. Here we show that Notch1 and its ligand Jagged1 are present at the synapse, and that Notch signaling in neurons occurs in response to synaptic activity. In addition, neuronal Notch signaling is positively regulated by Arc/Arg3.1, an activity-induced gene required for synaptic plasticity. In Arc/Arg3.1 mutant neurons, the proteolytic activation of Notch1 is disrupted both in vivo and in vitro. Conditional deletion of Notch1 in the postnatal hippocampus disrupted both long-term potentiation (LTP) and long-term depression (LTD), and led to deficits in learning and short-term memory. Thus, Notch signaling is dynamically regulated in response to neuronal activity, Arc/Arg3.1 is a context-dependent Notch regulator, and Notch1 is required for the synaptic plasticity that contributes to memory formation.