Consistent probabilistic outputs for protein function prediction

In predicting hierarchical protein function annotations, such as terms in the Gene Ontology (GO), the simplest approach makes predictions for each term independently. However, this approach has the unfortunate consequence that the predictor may assign to a single protein a set of terms that are inconsistent with one another; for example, the predictor may assign a specific GO term to a given protein ('purine nucleotide binding') but not assign the parent term ('nucleotide binding'). Such predictions are difficult to interpret. In this work, we focus on methods for calibrating and combining independent predictions to obtain a set of probabilistic predictions that are consistent with the topology of the ontology. We call this procedure 'reconciliation'. We begin with a baseline method for predicting GO terms from a collection of data types using an ensemble of discriminative classifiers. We apply the method to a previously described benchmark data set, and we demonstrate that the resulting predictions are frequently inconsistent with the topology of the GO. We then consider 11 distinct reconciliation methods: three heuristic methods; four variants of a Bayesian network; an extension of logistic regression to the structured case; and three novel projection methods - isotonic regression and two variants of a Kullback-Leibler projection method. We evaluate each method in three different modes - per term, per protein and joint - corresponding to three types of prediction tasks. Although the principal goal of reconciliation is interpretability, it is important to assess whether interpretability comes at a cost in terms of precision and recall. Indeed, we find that many apparently reasonable reconciliation methods yield reconciled probabilities with significantly lower precision than the original, unreconciled estimates. On the other hand, we find that isotonic regression usually performs better than the underlying, unreconciled method, and almost never performs worse; isotonic regression appears to be able to use the constraints from the GO network to its advantage. An exception to this rule is the high precision regime for joint evaluation, where Kullback-Leibler projection yields the best performance.     

Replication blocking lesions present a unique substrate for homologous recombination

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases.Retinal pigment epithelium (RPE) is a single neuroectodermal layer placed in the outermost part of the eye cup faced to photoreceptors.^{1, 2} Owing to its anatomical location and function, RPE is continuously exposed to potential cell damage caused by oxidative stress, specifically due to oxygen and nitrogen reactive species.³ This is probably one of the reasons why these cells are more resistant to oxidative stress.⁴ Oxidative stress is present as part of the pathophysiology in several retinal degenerations associated with blindness, for example, age-related macular degeneration,³ where RPE is considered a key factor for its development.⁵ Studies with the human-derived cell line ARPE-19 have proven to be very useful in the elucidation of the role of these cells in disease.Autophagy is a catabolic process aimed to degrade damaged organelles, proteins and cellular debris by engulfing them into a double membrane vesicle called the autophagosome and eliminating them by posterior fusion with the lysosome. Activation of macroautophagy, a form of autophagy, has been recently confirmed to be a primary response of ARPE-19 cells to stress.⁶ Furthermore, the two major functions of RPE, phagocytosis of the photoreceptor outer segments and visual cycle performance, have been linked to a noncanonical form of autophagy that is known as LC3 (microtubule-associated protein 1A/1B-light chain 3)-associated phagocytosis and is supposed to contribute to the normal supply of vitamin A and therefore to normal vision.^{7, 8}Despite its negative effects on health, ethanol (EtOH) is consumed daily worldwide, standing as one of the top public health challenges. EtOH induces morphological and physiological changes in the nervous tissue, and most of these changes may be attributed to reactive oxygen species (ROS), as they can be normalized or prevented by antioxidant treatments.^{9, 10, 11, 12, 13} Autophagy has been identified as cytoprotector in nervous and liver cells under EtOH-induced toxicity,^{14, 15} where it seems to degrade damaged organelles, including mitochondria. Recent findings support the idea that there is an increased mitochondrial stress and dysfunction in the RPE cells in AMD patients.^{16, 17} Oxidative-damaged mitochondria, a main source of ROS, seem to be removed by autophagy (known as mitophagy), in order to guarantee cell survival.¹⁸ As a matter of fact, deregulation of mitophagy has been implicated in several neurodegenerative diseases, such as Parkinson''s disease (PD), Alzheimer''s disease (AD) and Huntington''s disease (HD).Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress, and results in the generation of various bioactive compounds, among others 4-hydroxyalkenals (HAE). ROS-induced lipid peroxidation and the resulting HAE markedly contribute to the development and progression of different diseases.¹⁹ Specifically, 4-hydroxy-nonenal (4-HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of maladies.²⁰ It has been reported that 4-HNE induces apoptosis in ARPE-19 cells²¹ and its ability to form protein adducts, thus it seems to be a key factor in aggresome formation. Aggresome is a term referred to cytoplasmic perinuclear inclusion bodies formed by aggregated proteins.²² Indeed, the presence of aggresomes is a pathological hallmark of most neurodegenerative diseases, and 4-HNE seems to be involved in their formation in AD,²³ PD,²⁴ HD²⁵ and amyotrophic lateral sclerosis.²⁶ These aggresomes depend on the protein type to be cleared,^{27, 28} and their degradation by autophagy, known as aggrephagy, has been proposed to increase cell viability in neurodegeneration models.²⁹ Interestingly, 4-HNE aggregates have been also found in hepatic cells from alcoholic patients.^{30, 31, 32} Recent data provide no clear cut evidence of a link between PD risk and alcohol consumption with both positive³³ and negative³⁴ results.In this study, we report the cellular effects of EtOH on ARPE-19 cells and determine that EtOH exposure induces the formation of 4-HNE-aggresomes, together with other neurodegenerative hallmarks such as mitochondrial damage and autophagy activation. Considering the central role of RPE in retinal physiology and pathophysiology, and its neural origin, these findings render new insights into the mechanism of neurodegeneration caused by alcohol toxicity in retinal cells, and may contribute to the development of therapeutic strategies in several nervous and retinal diseases.     

The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin,?a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.     

A Simple Dynamic Model of Respiratory Pump

To study the interaction of forces that produce chest wall motion, we propose a model based on the lever system of Hillman and Finucane (J Appl Physiol 63(3):951–961, 1987) and introduce some dynamic properties of the respiratory system. The passive elements (rib cage and abdomen) are considered as elastic compartments linked to the open air via a resistive tube, an image of airways. The respiratory muscles (active) force is applied to both compartments. Parameters of the model are identified in using experimental data of airflow signal measured by pneumotachography and rib cage and abdomen signals measured by respiratory inductive plethysmography on eleven healthy volunteers in five conditions: at rest and with four level of added loads. A breath by breath analysis showed, whatever the individual and the condition are, that there are several breaths on which the airflow simulated by our model is well fitted to the airflow measured by pneumotachography as estimated by a determination coefficient R ² ≥ 0.70. This very simple model may well represent the behaviour of the chest wall and thus may be useful to interpret the relative motion of rib cage and abdomen during quiet breathing.     

Early endothelial progenitor cells in bone marrow are a biomarker of cell therapy success in patients with critical limb ischemia

Background aimsEndothelial progenitor cells (EPC) have been proposed for autologous angiogenic therapy. The objectives of this study were to quantify EPC in the peripheral blood and bone marrow mononuclear cells (BM-MNC) of patients with critical limb ischemia that had received BM-MNC as a cell therapy product, and to study the putative relationship between the presence of EPC and the process of neovascularization in toe or transmetatarsal amputation specimens.MethodsEarly and late endothelial progenitor cells (CFU-EC and ECFC) were cultivated and quantified according to published methods in peripheral blood and BM-MNC from patients with critical limb ischemia (CLI; n = 11) enrolled in the OPTIPEC trial (http://clinicaltrials.gov/ct2/show/NCT00377897) to receive BM-MNC as a cell therapy product.ResultsEight out of the 11 patients had undergone amputations. Three of the patients displayed a neoangiogenic process that was associated with a higher number of CFU-EC in BM-MNC, while CD3⁺, CFU-GM and CD34⁺ in BM-MNC, and EPC in peripheral blood, did not correlate with the appearance of newly formed vessels. As expected, circulating CFU-EC and ECFC counts were significantly lower in CLI patients compared with age-matched controls.ConclusionsIn patients with critical limb ischemia, EPC in peripheral blood were decreased compared with healthy individuals. However, in BM-MNC we found that relative numbers of CFU-EC could be used as an indicator to discriminate patients with neoangiogenic processes. These results need to be confirmed in a randomized study.     

Design and synthesis of bridged gamma-lactams as analogues of beta-lactam antibiotics

Anti-Bredt bridged bicyclo[3.2.1] gamma-lactams were designed as inhibitors of penicillin binding proteins (PBPs). The compounds were prepared by a carbenoid insertion into a lactam N-H bond. Their weak antibacterial activity could either be explained by a poor chemical stability or by unfavorable steric interactions of the methylene bridge of the gamma-lactam with the targeted enzymes.