Dynamic Dependence on ATR and ATM for Double-Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.     

The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1–40 and Aβ1–42 are the dominant forms. The fibril architectures of Aβ1–40 and Aβ1–42 differ and Aβ1–42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1–42 can be cross-templated and incorporated into the ends of Aβ1–40 fibrils, while incorporation of Aβ1–40 monomers into Aβ1–42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1–40 to incorporate into the ends of Aβ1–42 fibrils and the capacity of Aβ1–42 monomers to adopt the properties of Aβ1–40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1–42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1–40 from adopting the fibrillar properties of Aβ1–42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.     

An importin alpha homolog, MOS6, plays an important role in plant innate immunity

Plant disease resistance is the consequence of an innate defense mechanism mediated by Resistance (R) genes [1]. The conserved structure of one class of R protein is reminiscent of Toll-like receptors (TLRs) and Nucleotide binding oligomerization domain (NOD) proteins-immune-response perception modules in animal cells [2, 3, and 4]. The Arabidopsis snc1 (suppressor of npr1-1, constitutive, 1) mutant contains a mutation in a TIR-NBS-LRR-type of R gene that renders resistance responses constitutively active without interaction with pathogens [5]. Few components of the downstream signaling network activated by snc1 are known. To search for regulators of R-gene-mediated resistance, we screened for genetic suppressors of snc1. Three alleles of the mutant mos6 (modifier of snc1, 6) partially suppressed constitutive-resistance responses and immunity to virulent pathogens in snc1. Furthermore, the mos6-1 single mutant exhibited enhanced disease susceptibility to a virulent oomycete pathogen. MOS6, identified by positional cloning, encodes importin alpha3, one of eight alpha importins in Arabidopsis [6]. alpha importins mediate the import of specific proteins across the nuclear envelope. We previously reported that MOS3, a protein homologous to human nucleoporin 96, is required for constitutive resistance in snc1 [7]. Our data highlight an essential role for nucleo-cytoplasmic trafficking, especially protein import, in plant innate immunity.     

Requirement of Tyr-992 and Tyr-1173 in phosphorylation of the epidermal growth factor receptor by ionizing radiation and modulation by SHP2

The epidermal growth factor receptor (EGFR) is activated by ionizing radiation (IR) in many human carcinomas, mediating a cytoprotective response and subsequent radioresistance. The underlying molecular mechanisms remain to be understood, and we propose here a specific role for the Tyr-992 residue of EGFR and examine its regulation by the phosphatase, SHP2. The -fold increase in phosphorylation of Tyr-992 in response to IR is twice that seen with ligand (EGF) binding. Mutation of Tyr-992 blocked completely IR-induced EGFR phosphorylation and reduced activation of the downstream signaling molecule, phospholipase Cgamma. IR has previously been demonstrated to inhibit activity of protein-tyrosine phosphatases. Following protein-tyrosine phosphatase inhibition by sodium vanadate both EGFR expressing Chinese hamster ovary (CHO) and A431 exhibited up to an 8-fold increase in the basal level of Tyr-992 phosphorylation, significantly higher than that seen with Tyr-1173, Tyr-1068, and total EGFR Tyr. CHO cells expressing a SHP2 mutant also demonstrated up to an 8-fold increase in the basal level of Tyr-992 phosphorylation. In this study we show the unique association of SHP2 with EGFR in response to IR, with up to a 2.5-fold increase in the direct association of endogenous SHP2 with EGFR-wt in response to 2 gray of IR in both CHO and A431 cells. Mutation of Tyr-992 abolished this response. In conclusion we have identified several differentially activated Tyr residues, one of which is not only more sensitive to activation by IR, translating into differential activation of downstream signaling, but uniquely modulated by the phosphatase SHP2.     

Assessments of Thioridazine as a Helper Compound to Dicloxacillin against Methicillin-Resistant Staphylococcus aureus: In Vivo Trials in a Mouse Peritonitis Model

Introduction

The rise in antimicrobial resistance is a major global concern and requires new treatment strategies. The use of helper compounds, such as thioridazine (TDZ), an antipsychotic drug, in combination with traditional antibiotics must be investigated.

Objectives

The aim of this study was to investigate the efficacy of TDZ as a helper compound for dicloxacillin (DCX) against methicillin-resistant Staphylococcus aureus (MRSA) in vivo, and compare the combination treatment of DCX+TDZ with vancomycin (VAN).

Methods

Mice were inoculated with an intraperitoneal (IP) injection of MRSA (10⁸ CFU) and treated in a 12-hour cycle for 48 hours. By termination, bacterial quantities in a peritoneal flush, spleen and kidneys were obtained. In the main trial the drugs were administered subcutaneously in five treatment groups: 1) DCX, 2) TDZ, 3) DCX+TDZ, 4) VAN, 5) SALINE. Additional smaller studies with IP administration and higher subcutaneous dosages (×1.5 and ×4) of the drugs were subsequently performed.

Results

In the main trial no significant differences were found between DCX+TDZ and DCX or TDZ alone (p≥0.121–0.999). VAN performed significantly better than DCX+TDZ on all bacteriological endpoints (p<0.001). Higher subcutaneous dosages of DCX and TDZ improved the antibacterial efficacy, but the combination treatment was still not significantly better than monotherapy. IP drug administration of DCX+TDZ revealed a significantly better antibacterial effect than DCX or TDZ alone (p<0.001) but not significantly different from VAN (p>0.999).

Conclusion

In conclusion, TDZ did not prove to be a viable helper compound for dicloxacillin against MRSA in subcutaneous systemic treatment. However, IP-administration of DCX+TDZ, directly at the infection site resulted in a synergetic effect, with efficacy comparable to that of VAN.     

Microfibrillar-Associated Protein 4: A Potential Biomarker for Screening for Liver Fibrosis in a Mixed Patient Cohort

MethodspMFAP4 was measured in samples from 351 drug users attending treatment centres and from 248 acutely hospitalized medical patients with mixed diagnoses. Linear and logistic multivariate regression analyses were performed and nonparametric receiver operating characteristic-curves for cirrhosis were used to estimate cut-off points for pMFAP4. Univariate and subgroup analyses were performed using non-parametric methods.ResultspMFAP4 increased significantly with liver fibrosis score. pMFAP4 was significantly associated with chronic viral infection in the drug users and with transient elastography in both cohorts. In the mixed patient cohort, pMFAP4 was significantly increased among patients with a previous diagnosis of liver disease or congestive heart failure compared to patients with other diagnoses.ConclusionspMFAP4 has the potential to be used as an outreach-screening tool for liver fibrosis in drug users and in mixed medical patients. pMFAP4 level is positively associated with transient elastography, but additional studies are warranted to validate the possible use of pMFAP4 in larger cohorts and in combination with transient elastography.     

Increased Reward-Related Behaviors during Sleep and Wakefulness in Sleepwalking and Idiopathic Nightmares

Background

We previously suggested that abnormal sleep behaviors, i.e., as found in parasomnias, may often be the expression of increased activity of the reward system during sleep. Because nightmares and sleepwalking predominate during REM and NREM sleep respectively, we tested here whether exploratory excitability, a waking personality trait reflecting high activity within the mesolimbic dopaminergic (ML-DA) system, may be associated with specific changes in REM and NREM sleep patterns in these two sleep disorders.

Methods

Twenty-four unmedicated patients with parasomnia (12 with chronic sleepwalking and 12 with idiopathic nightmares) and no psychiatric comorbidities were studied. Each patient spent one night of sleep monitored by polysomnography. The Temperament and Character Inventory (TCI) was administered to all patients and healthy controls from the Geneva population (n = 293).

Results

Sleepwalkers were more anxious than patients with idiopathic nightmares (Spielberger Trait anxiety/STAI-T), but the patient groups did not differ on any personality dimension as estimated by the TCI. Compared to controls, parasomnia patients (sleepwalkers together with patients with idiopathic nightmares) scored higher on the Novelty Seeking (NS) TCI scale and in particular on the exploratory excitability/curiosity (NS1) subscale, and lower on the Self-directedness (SD) TCI scale, suggesting a general increase in reward sensitivity and impulsivity. Furthermore, parasomnia patients tended to worry about social separation persistently, as indicated by greater anticipatory worry (HA1) and dependence on social attachment (RD3). Moreover, exploratory excitability (NS1) correlated positively with the severity of parasomnia (i.e., the frequency of self-reported occurrences of nightmares and sleepwalking), and with time spent in REM sleep in patients with nightmares.

Conclusions

These results suggest that patients with parasomnia might share common waking personality traits associated to reward-related brain functions. They also provide further support to the notion that reward-seeking networks are active during human sleep.     

RGB marking facilitates multicolor clonal cell tracking

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.     

An introduction to membrane proteins

α-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics.     

Cytochrome c-554 from Methylosinus trichosporium OB3b; a protein that belongs to the cytochrome c2 family and exhibits a HALS-Type EPR signal

A small soluble cytochrome c-554 purified from Methylosinus trichosporium OB3b has been purified and analyzed by amino acid sequencing, mass spectrometry, visible, CD and EPR spectroscopies. It is found to be a mono heme protein with a characteristic cytochrome c fold, thus fitting into the class of cytochrome c(2), which is the bacterial homologue of mitochondrial cytochrome c. The heme iron has a Histidine/Methionine axial ligation and exhibits a highly anisotropic/axial low spin (HALS) EPR signal, with a g(max) at 3.40, and ligand field parameters V/ξ = 0.99, Δ/ξ = 4.57. This gives the rhombicity V/Δ = 0.22. The structural basis for this HALS EPR signal in Histidine/Methionine ligated hemes is not resolved. The ligand field parameters observed for cytochrome c-554 fits the observed pattern for other cytochromes with similar ligation and EPR behaviour.