Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.     

A stress surveillance system based on calcium and nitric oxide in marine diatoms

Diatoms are an important group of eukaryotic phytoplankton, responsible for about 20% of global primary productivity. Study of the functional role of chemical signaling within phytoplankton assemblages is still in its infancy although recent reports in diatoms suggest the existence of chemical-based defense strategies. Here, we demonstrate how the accurate perception of diatom-derived reactive aldehydes can determine cell fate in diatoms. In particular, the aldehyde (2E,4E/Z)-decadienal (DD) can trigger intracellular calcium transients and the generation of nitric oxide (NO) by a calcium-dependent NO synthase-like activity, which results in cell death. However, pretreatment of cells with sublethal doses of aldehyde can induce resistance to subsequent lethal doses, which is reflected in an altered calcium signature and kinetics of NO production. We also present evidence for a DD–derived NO-based intercellular signaling system for the perception of stressed bystander cells. Based on these findings, we propose the existence of a sophisticated stress surveillance system in diatoms, which has important implications for understanding the cellular mechanisms responsible for acclimation versus death during phytoplankton bloom successions.     

Stern-judging: A simple, successful norm which promotes cooperation under indirect reciprocity

We study the evolution of cooperation under indirect reciprocity, believed to constitute the biological basis of morality. We employ an evolutionary game theoretical model of multilevel selection, and show that natural selection and mutation lead to the emergence of a robust and simple social norm, which we call stern-judging. Under stern-judging, helping a good individual or refusing help to a bad individual leads to a good reputation, whereas refusing help to a good individual or helping a bad one leads to a bad reputation. Similarly for tit-for-tat and win-stay-lose-shift, the simplest ubiquitous strategies in direct reciprocity, the lack of ambiguity of stern-judging, where implacable punishment is compensated by prompt forgiving, supports the idea that simplicity is often associated with evolutionary success.     

An environment for relation mining over richly annotated corpora: the case of GENIA

Background

The biomedical domain is witnessing a rapid growth of the amount of published scientific results, which makes it increasingly difficult to filter the core information. There is a real need for support tools that 'digest' the published results and extract the most important information.

Results

We describe and evaluate an environment supporting the extraction of domain-specific relations, such as protein-protein interactions, from a richly-annotated corpus. We use full, deep-linguistic parsing and manually created, versatile patterns, expressing a large set of syntactic alternations, plus semantic ontology information.

Conclusion

The experiments show that our approach described is capable of delivering high-precision results, while maintaining sufficient levels of recall. The high level of abstraction of the rules used by the system, which are considerably more powerful and versatile than finite-state approaches, allows speedy interactive development and validation.

     

Bright oligothiophene N-succinimidyl esters for efficient fluorescent labeling of proteins and oligonucleotides

The synthesis of multicolor fluorescent oligothiophene N-succinimidyl esters (TSEs) is reported, and their optical properties are discussed with the aid of ab initio calculations. The esters were coupled to proteins and to 3'-amino-modified oligonucleotides in mild conditions and with similar modalities. A comparative study of the bioconjugate of IgG1 anti-CD3 antibody labeled with a blue fluorescent TSE and with fluorescein isothiocyanate (FITC) is reported, showing that the former achieves higher photoluminescence intensity and optical stability than the latter. Fluorescence resonance energy transfer experiments with TSE-labeled oligonucleotides and examples of cellular imaging via TSE-labeled proteins are reported.     

Single tube genotyping of GSTM1, GSTT1 and TP53 polymorphisms by multiplex PCR.

Glutathione S-transferases (GST) are enzymes involved in the conjugation of a number of human carcinogens, while p53 tumour suppressor gene is the most frequently mutated gene identified till now in human neoplasias. Typically, GSTM1 and GSTT1 genotyping are performed together, with several different protocol described and sometimes with the risk of misclassification due to "false negative", depending on the internal positive control employed. Here, we report a modification of the classical multiplex polymerase chain reaction (PCR) method, allowing the genotyping of GSTM1, GSTT1, together with a polymorphism within the intron 3 of TP53 tumour suppressor gene (a 16 base pairs (bp) duplication) in a single tube, with an appropriate internal positive control. To test the applicability of the method, the frequencies of the deleted alleles of GSTM1 and GSTT1 (null genotypes), and the 16 bp duplication of TP53 gene were assayed in a series of Caucasian DNA samples.     

Intensification of the aerobic bioremediation of an actual site soil historically contaminated by polychlorinated biphenyls (PCBs) through bioaugmentation with a non acclimated, complex source of microorganisms

Background  

The biotreatability of actual-site polychlorinated biphenyl (PCB)-contaminated soils is often limited by their poor content of autochthonous pollutant-degrading microorganisms. In such cases, inoculation might be the solution for a successful bioremediation. Some pure and mixed cultures of characterized PCB degrading bacteria have been tested to this purpose. However, several failures have been recorded mostly due to the inability of inoculated microbes to compete with autochthonous microflora and to face the toxicity and the scarcity of nutrients occurring in the contaminated biotope. Complex microbial systems, such as compost or sludge, normally consisting of a large variety of robust microorganisms and essential nutrients, would have better chances to succeed in colonizing degraded contaminated soils. However, such sources of microorganisms have been poorly applied in soil bioremediation and in particular in the biotreatment of soil with PCBs. Thus, in this study the effects of Enzyveba, i.e. a consortium of non-adapted microorganisms developed from composted material, on the slurry- and solid-phase aerobic bioremediation of an actual-site, aged PCB-contaminated soil were studied.     

Photo-cross-linked hydrogels with polysaccharide-poly(amino acid) structure: new biomaterials for pharmaceutical applications

The aim of this work has been the preparation and characterization of novel hydrogels with polysaccharide-poly(amino acid) structure having suitable physicochemical properties for pharmaceutical applications. In the first step, hyaluronic acid (HA) and alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) have been derivatized with methacrylic anhydride (AMA), thus obtaining HA-AMA and PHM derivatives, respectively. In the second step, aqueous solutions of both these derivatives have been irradiated at 313 nm to obtain chemical hydrogels. The hydrogel obtained by irradiating for 15 min an aqueous solution containing 4% w/v of HA-AMA and 4% w/v of PHM resulted in the highest yield. Its swelling ability was dependent on the pH and nature of the external medium. Besides, this hydrogel undergoes a partial hydrolysis, especially in the presence of enzymes, such as esterase or hyaluronidase, but the entity of this degradation is lower than that observed for a hydrogel based on HA-AMA alone. The ability of this hydrogel to entrap drug molecules has been evaluated by using thrombin as a model drug. In vitro release studies and a platelet aggregation test demonstrated that the HA-AMA/PHM hydrogel is able to release thrombin in the active form, thus suggesting its suitability for the treatment of hemorrhages.     

Low-dose enalapril in the treatment of surgical cutaneous hypertrophic scar and keloid--two case reports and literature review

Hypertrophic scars and keloids are 2 forms of excessive cutaneous scarring that occur in predisposed individuals. The healing process varies greatly among patients, and the risk of a bad scar evolution is unpredictable. Keloids create disfiguring scars with associated erythema and pain or pruritus or restricted range of motion, and are a major cause of morbidity. A fortuitous observation was made by the first author of this study who, at age 54, developed an erythematous and painful postsurgical abdominal keloid scar after undergoing left colectomy for colon adenocarcinoma. Four months later, after treatment with low-dose enalapril (10 mg, once a day) for mild arterial hypertension, her keloid scar rapidly improved and she eventually made a complete recovery. second case involved a 70-year-old female with diabetes who was affected by a long-standing postsurgical abdominal keloid scar of 2 years' duration. She was intentionally treated with the same low dose of enalapril, and, after 6 months of therapy, the bad scar showed marked improvement. We conducted an exhaustive search of the literature pertaining to the wound healing process, specifically to determine whether angiotensin-converting enzyme (ACE) inhibitors have a healing effect on wounds. ACE inhibitors are known to induce reduction of left ventricular collagen content and to attenuate remodeling during the postinfarctual period (thus improving ventricular function), and they have been shown to exert a pulmonary antifibrotic effect. After conducting this literature search, it became apparent that no data on cutaneous scars and ACE inhibitors are available. During the posttraumatic or postoperative stage, it is useful to achieve the best possible aesthetic results and to decrease the risk of a disfiguring keloid scar, thereby avoiding revision surgery; to this purpose, an early treatment with a low dose of enalapril is a possible solution, even if further confirmatory observations are needed.