Proteome Data Improves Protein Function Prediction in the Interactome of Helicobacter pylori.

Helicobacter pylori is a common pathogen that is estimated to infect half of the human population, causing several diseases such as duodenal ulcer. Despite one of the first pathogens to be sequenced, its proteome remains poorly characterized as about one-third of its proteins have no functional annotation. Here, we integrate and analyze known protein interactions with proteomic and genomic data from different sources. We find that proteins with similar abundances tend to interact. Such an observation is accompanied by a trend of interactions to appear between proteins of similar functions, although some show marked cross-talk to others. Protein function prediction with protein interactions is significantly improved when interactions from other bacteria are included in our network, allowing us to obtain putative functions of more than 300 poorly or previously uncharacterized proteins. Proteins that are critical for the topological controllability of the underlying network are significantly enriched with genes that are up-regulated in the spiral compared with the coccoid form of H. pylori Determining their evolutionary conservation, we present evidence that 80 protein complexes are identical in composition with their counterparts in Escherichia coli, while 85 are partially conserved and 120 complexes are completely absent. Furthermore, we determine network clusters that coincide with related functions, gene essentiality, genetic context, cellular localization, and gene expression in different cellular states.

A second-generation protein-protein interaction network of Helicobacter pylori.

Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

RsfA (YbeB) proteins are conserved ribosomal silencing factors.

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.

Differences between unipolar and bipolar I depression in the quantitative analysis of glutamic acid decarboxylase-immunoreactive neuropil.

Alterations in GABAergic neurotransmission are assumed to play a crucial role in the pathophysiology of mood disorders. Glutamic acid decarboxylase (GAD) is the key enzyme in GABA synthesis. This study aimed to differentiate between unipolar and bipolar I depression using quantitative evaluation of GAD-immunoreactive (GAD-ir) neuropil in several brain regions known to be involved in the pathophysiology of mood disorders. Immunohistochemical staining of GAD 65/67 was performed in the orbitofrontal, anterior cingulate and dorsolateral prefrontal cortex (DLPFC), the entorhinal cortex, the hippocampal formation and the medial dorsal and lateral dorsal (LD) thalamic nuclei, with a quantitative densitometric analysis of GAD-ir neuropil. The study was performed on paraffin-embedded brains from 9 unipolar and 12 bipolar I depressed patients (8 and 6 suicidal patients, respectively) and 18 matched controls. In unipolar patients, compared with controls, only the increased relative density of GAD-ir neuropil in the right LD was different from the previous results in depressed suicides from the same cohort (Gos et al. in J Affect Disord 113:45-55, 2009). On the other hand, the left DLPFC was the only area where a significant decrease was observed, specific for bipolar I depression. Significant differences between both diagnostic groups were found in these regions. By revealing abnormalities in the relative density of GAD-ir neuropil in brain structures, our study suggests a diathesis of the GABAergic system in mood disorders, which may differentiate the pathophysiology of unipolar from that of bipolar I depression.

The proteome and interactome of Streptococcus pneumoniae phage Cp-1.

Mass spectrometry analysis of Streptococcus pneumoniae bacteriophage Cp-1 identified a total of 12 proteins, and proteome-wide yeast two-hybrid screens revealed 17 binary interactions mainly among these structural proteins. On the basis of the resulting linkage map, we suggest an improved structural model of the Cp-1 virion.

Genome annotation and intraviral interactome for the Streptococcus pneumoniae virulent phage Dp-1.

Streptococcus pneumoniae causes several diseases, including pneumonia, septicemia, and meningitis. Phage Dp-1 is one of the very few isolated virulent S. pneumoniae bacteriophages, but only a partial characterization is currently available. Here, we confirmed that Dp-1 belongs to the family Siphoviridae. Then, we determined its complete genomic sequence of 56,506 bp. It encodes 72 open reading frames, of which 44 have been assigned a function. We have identified putative promoters, Rho-independent terminators, and several genomic clusters. We provide evidence that Dp-1 may be using a novel DNA replication system as well as redirecting host protein synthesis through queuosine-containing tRNAs. Liquid chromatography-mass spectrometry analysis of purified phage Dp-1 particles identified at least eight structural proteins. Finally, using comprehensive yeast two-hybrid screens, we identified 156 phage protein interactions, and this intraviral interactome was used to propose a structural model of Dp-1.

Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome.

BACKGROUND: Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. RESULTS: We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all approximately 4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About approximately 20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. CONCLUSIONS: Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays.

The potential use of the eye temperature decrease in determining the time of death in the early postmortem period: studies in pigs.

The experiment was performed in pigs during which the postmortem body temperature was continuously measured. The study was focused on a period of time starting 0.5 hours after the animals' termination and consisted in computer recording of the cooling process of the eyeball (vitreous humor), orbit soft tissues, muscles, and the rectum. The experiment revealed the absence of a plateau phase of temperature decrease in eyeball and orbit soft tissues contrary to muscles and rectum, what demonstrates that temperature measurements in the eye could become an alternative method of determining the time of death during the initial phase of postmortem cooling. During this early postmortem period determination of the time of death based on measurements of the rectum or muscles temperatures due to existing plateau is not possible.

A preliminary study for identifying olfactory markers of fear in the rat.

In stressful situations, many animals release alarm pheromones to warn conspecifics of impending danger. The authors sought to establish experimental conditions for a larger study aimed at identifying alarm pheromones emitted by the rat. They placed rats in a specially designed chamber and exposed them to aversive tactile, visual and acoustic stimuli over the course of a few days. The researchers observed rats' behavior and analyzed air samples taken from their immediate environment under the following conditions: (i) when rats were unstressed; (ii) immediately after rats were exposed to aversive stimuli; and (iii) when rats were left alone in the chamber after being conditioned to fear imminent aversive stimuli. Stressed rats emitted several substances that are known to function as alarm pheromones in insects. When previously unstressed control rats were exposed to these same substances, they had a distinct behavioral fear response.

The Escherichia coli protein YjjG is a house-cleaning nucleotidase in vivo.

House-cleaning enzymes protect cells from the adverse effects of noncanonical metabolic chemical compounds. The Escherichia coli nucleotide phosphatase YjjG (B4374, JW4336) functions as a house-cleaning phosphatase in vivo. YjjG protects the cell against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine (5-FdUridine), 5-fluorouridine, 5-fluoroorotic acid (5-FOA), 5-fluorouracil, and 5-aza-2'-deoxycytidine. YjjG prevents the incorporation of potentially mutagenic nucleotides into DNA as shown for 5-bromo-2'-deoxyuridine (BrdU). Its enzymatic activity in vitro towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP) is higher than towards canonical thymidine monophosphate (dTMP). The closest homolog in humans, HDHD4, does not show a protective effect against noncanonical nucleotides, excluding an involvement of HDHD4 in resistance against noncanonical nucleotides used for cancer chemotherapy. The substrate spectrum of YjjG suggests that its in vivo substrates are noncanonical pyrimidine derivatives, which might also include oxidized nucleobases such as 5-formyluracil and 5-hydroxyuracil.

[Estimation of the time of death based on the measurements of the eye temperature in comparison with other body sites]. / Okreslanie czasu zgonu na podstawie pomiaru temperatury oka wobec innych miejsc ciala.

A systematic two-stage study was conducted in pigs to verify the models of postmortem body temperature decrease currently employed in forensic medicine. During the investigations, temperature recordings were performed in four body sites (eyeballs, orbit soft tissues, muscles and rectums). The results of the study support the possible use of the eyeball and also the orbit soft tissues as temperature measuring sites at the early phase after death; they have narrowed the significance of rectum temperature measurements to the late stage of postmortem body temperature decrease, shown insignificant correlations between the body weight and the temperature decrease rate constant and illustrated the functional increase of the time of death estimation error as the body cools, expressed in the distinct tendency to overestimate the calculated time of death as compared to the actual one. In the second stage of the experiment, a lack of a plateau phase was demonstrated, at least from 30 min post mortem. It was also found that in the very early post mortem period, the kinetics of cooling of all the body sites studied was better described by the two-exponential model than the single exponential one. The study also showed that the weak airflow present in the experimental conditions did not practically affect the course of cooling of the investigated body sites. Eyeball temperature measurements with an infra-red laser thermometer performed during the experiment proved to be of no use for determination of the time of death. The experiments allowed for defining the so far unreported value of physiological temperature of pig eyeball as 38 degrees C.

The interactome of Streptococcus pneumoniae and its bacteriophages show highly specific patterns of interactions among bacteria and their phages.

Although an abundance of bacteriophages exists, little is known about interactions between their proteins and those of their bacterial hosts. Here, we experimentally determined the phage-host interactomes of the phages Dp-1 and Cp-1 and their underlying protein interaction network in the host Streptococcus pneumoniae. We compared our results to the interaction patterns of E. coli phages lambda and T7. Dp-1 and Cp-1 target highly connected host proteins, occupy central network positions, and reach many protein clusters through the interactions of their targets. In turn, lambda and T7 targets cluster to conserved and essential proteins in E. coli, while such patterns were largely absent in S. pneumoniae. Furthermore, targets in E. coli were mutually strongly intertwined, while targets of Dp-1 and Cp-1 were strongly connected through essential and orthologous proteins in their immediate network vicinity. In both phage-host systems, the impact of phages on their protein targets appears to extend from their network neighbors, since proteins that interact with phage targets were located in central network positions, have a strong topologically disruptive effect and touch complexes with high functional heterogeneity. Such observations suggest that the phages, biological impact is accomplished through a surprisingly limited topological reach of their targets.

The estimation of stature on the basis of measurements of the femur.

The study aims to establish the relationship between body height and the greatest length of the femur. The motive for undertaking these examinations was the lack in the literature of studies that allow -- with reference to the current population of Central Europe -- the reconstruction of height while alive based on measurements of a skeleton. It was decided to examine isolated bones from human remains undergoing forensic autopsy, and belonging to individuals of both sexes whose growth processes had stopped. Examinations were conducted on 91 human bodies from the contemporary Polish population. The research points to a very close relationship between the length of a dead body and the measured greatest length of the femur. This relationship was expressed in nine coefficients of correlation calculated for both sexes.

[Issues related to the identification process of catastrophes victims]. / Problematyka identyfikacji ofiar katastrof.

Catastrophes on an international scale with an increasingly pronounced terrorist factor (for example, the terrorist attacks in the United States of America on September 11, 2001) are occurring with increasing frequency. This fact compels the international forensic medical community--especially at the present moment--to undertake coordinated and rapid measures in the area, closely related to our profession, of issues connected with problems of identifying the victims of catastrophes. To date organizational issues have not been harmonized to the required at an international level degree. The subject has been raised frequently at congresses and symposiums of forensic medicine. The real and present danger of catastrophes at a national and regional level demands the adoption of rapid organizational activities at these levels too. Because the victims of such catastrophes are often of various nationalities, these activities also demand urgent organizational harmonization in keeping with established international norms. The following article addresses this issue. It contains propositions in outline with the aim of presenting them to discussion. These suggestions refer solely to organizational activities on the regional, national, and international levels, connected with the issue of identification in the broad sense of the word.

[Controversies concerning the comma. The voice in the discussion]. / Kontrowersje wokól przecinka. Glos w dyskusji.

In the article, a case is presented, which was assessed in accordance with the Constitutional Tribunal sentence of July 7th, 2003. Due to an essential change in the legal system entailing a return to the contents of art. 156 section 1 p. 2 of the Penal Code resolved on June 6th, 1997, this sentence caused the forming of the opinions which are medically illogical and contradictory to medico-legal experience.

[Retinal hemorrhages as a case for shaking trauma. Case report]. / Wylewy krwawe w siatkówkach jako istotny dowód wskazujacy na okolicznosci ich powstania.

An interesting shaking trauma case was reported. A detailed analysis of the pattern of injuries and their progress allowed to reconstruct the mechanism and time point of cerebral lesions in a 6 week old infant who survived the impact. The necessity of postmortem investigation of the fundus and cranial nerves in fatal cases of shaking trauma is stressed.

["Alarm substances" as biological markers indicating emotional state before death]. / Substancje alarmowe jako wykladnik stanu emocjonalnego poprzedzajacego zgon.

This scientific project presents the results of preliminary examinations aimed to identify alarm pheromones secreted by mammals. Wistar male rats were used for the experiment. Animals were treated by aversive sensoric stimuli and fear conditioning procedures. The animals' behaviour was registered. Quantitative and qualitative analysis of air samples taken from their environment was conducted with the use of GC-FID and GC-MSD technics. In the rhinocephalon structures (olfactory bulb, olfactory tract) the concentration of glutamate was measured. During the progress of the experiment increasing behavioral reactions of anxiety were observed in the rats. In their atmosphere organic compounds were identified. Some of them can be considered to be alarm substances. In the examined structures of the rhinocephalon on increased concentration of glutamate in each individual was revealed.

The EHEC-host interactome reveals novel targets for the translocated intimin receptor.

Enterohemorrhagic E. coli (EHEC) manipulate their human host through at least 39 effector proteins which hijack host processes through direct protein-protein interactions (PPIs). To identify their protein targets in the host cells, we performed yeast two-hybrid screens, allowing us to find 48 high-confidence protein-protein interactions between 15 EHEC effectors and 47 human host proteins. In comparison to other bacteria and viruses we found that EHEC effectors bind more frequently to hub proteins as well as to proteins that participate in a higher number of protein complexes. The data set includes six new interactions that involve the translocated intimin receptor (TIR), namely HPCAL1, HPCAL4, NCALD, ARRB1, PDE6D, and STK16. We compared these TIR interactions in EHEC and enteropathogenic E. coli (EPEC) and found that five interactions were conserved. Notably, the conserved interactions included those of serine/threonine kinase 16 (STK16), hippocalcin-like 1 (HPCAL1) as well as neurocalcin-delta (NCALD). These proteins co-localize with the infection sites of EPEC. Furthermore, our results suggest putative functions of poorly characterized effectors (EspJ, EspY1). In particular, we observed that EspJ is connected to the microtubule system while EspY1 appears to be involved in apoptosis/cell cycle regulation.

The binary protein-protein interaction landscape of Escherichia coli.

Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (∼70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, which approximately doubles the number of known binary PPIs in E. coli. Integration of binary PPI and genetic-interaction data revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that we could map in multiprotein complexes were informative regarding internal topology of complexes and indicated that interactions in complexes are substantially more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily important model microbe.

Lightning strike as probable cause of death and determining identity based on the examination of skeletal remains.

In this article, a case of examining an incomplete human skeleton found in a forest is presented. Based on the assessment of posttraumatic lesions in the skull bones, the cause of death of a man who had died 14 years prior to the examination was determined with high probability to be due to a lightning strike. Moreover, the rare pathological lesions within the skeleton, such as a healed fracture of the humerus and rheumatoid malformations within the hand, in reference to preserved medical records, have also allowed to identify the deceased. Most noteworthy points in this case are, however, initially difficult to comprehend, posttraumatic lesions within the bones of the skull such as carbonization and fracture of the vertex. Their origin can be explained by a lightning strike as the most probable cause of death. Thus, the presented case demonstrates probable cause of death and identity can be established many years after death, based on skeletal remains.