Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

Lipid anchors are common post-translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C-termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP-Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the structural basis of plant REP-Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule-specific, and in other cases they are common for a subset of Rabs. In particular, the C-terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C-terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

Differential diagnosis of a calcified cyst found in an 18th century female burial site at St. Nicholas Church cemetery (Libkovice, Czechia).

During archaeological excavations in burial sites, sometimes stoned organic objects are found, in addition to human remains. Those objects might be of a different origin, depending on various factors influencing members of a community (i.e. diseases, trauma), which provides information about their living conditions. The St. Nicholas Church archaeological site (Libkovice, Czechia) in the 18th century horizon of the cemetery, yielded a maturus-senilis female skeleton with a stone object in the left iliac fossa. This object was an oviform cyst-like rough structure, measuring 54 mm in length, 35 mm in maximum diameter and 0.2-0.7 mm shell thickness. Within the object there were small fetal bones (long bones, i.e. femur and two tibias, two scapulas, three ribs, vertebrae and other tiny bone fragments). Methods utilized to analyze the outer and inner surface morphology of the cyst and its inside, included: X-ray, CT imaging, SEM, histological staining and EDS. The EDS analysis revealed the presence of primarily oxygen, calcium and phosphorus in bone samples, and oxygen and silicon, in stone shell. Based on the length of the femur (20.2 mm) and tibia (16 mm) shafts, the fetal age was determined as being in the 15-18 week of pregnancy. The differential diagnosis was conducted, including for the three most probable cases: fetiform teratoma (FT), fetus-in-fetu (FIF) and lithopedion. The possibility of fetiform teratoma was discounted due to the presence of an anatomically correct spine, long bones and the proportions of the find. Although the low calcium content in the shell (2.3% atom mass), the lack of skull bones and the better developed lower limbs indicate fetus-in-fetu rather than lithopedion, the analyses results are unable to conclusively identify the object under one of these two categories since there are insufficient such cases in excavation material with which to draw comparison.

A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.

Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species. Despite only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologues and paralogues. We propose that Rpi-amr1 gene family diversity assists detection of diverse paralogues and alleles of the recognized effector, facilitating durable resistance against P. infestans.

CBCT for estimation of the cemento-enamel junction and crestal bone of anterior teeth.

The aim of the study is to evaluate the usefulness of cone-beam computed tomography (CBCT) in the assessment of the relationship between the cemento-enamel junction (CEJ) and bone crest of the anterior mandibular cortex. The study population comprised 39 males and 61 females, aged 18-71. A GENDEX GXCB-500 machine, i-CAT Vision and CorelDraw 9 software were used. The distances between the CEJ and bone crest at buccal and lingual sides of six anterior mandibular teeth were measured. Descriptive statistical methods, Student's t-test and ANOVA were used. The mean distance between the bone crest and CEJ was 2.32 mm ± 0.78 mm at the buccal and 2.52 mm ± 0.85 mm at the lingual side. It was found that in males aged over 50 years, the mean distance at the buccal side was 2.84 mm ± 0.79 mm and was significantly higher than in males aged 49 and less - 2.08 mm ± 0.41 mm. The mean distance at the lingual side was 3.28 mm ± 1.08 mm and was significantly lower in the age group of 49 years and less - 2.10 mm ± 0.41 mm. CBCT allows determining the distance between the CEJ and crestal bone margin at buccal and lingual sides. The data provide crucial information for planning orthodontic treatment, implant placement and periodontal therapy.

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.

The NLR-Annotator Tool Enables Annotation of the Intracellular Immune Receptor Repertoire.

Disease resistance genes encoding nucleotide-binding and leucine-rich repeat (NLR) intracellular immune receptor proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR-encoding genes. The availability of the hexaploid wheat (Triticum aestivum) cultivar Chinese Spring reference genome allows a detailed study of its NLR complement. However, low NLR expression and high intrafamily sequence homology hinder their accurate annotation. Here, we developed NLR-Annotator, a software tool for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. We applied our tool to wheat and combined it with a transcript-validated subset of genes from the reference gene annotation to characterize the structure, phylogeny, and expression profile of the NLR gene family. We detected 3,400 full-length NLR loci, of which 1,560 were confirmed as expressed genes with intact open reading frames. NLRs with integrated domains mostly group in specific subclades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains, suggesting a paired helper function. Most NLRs (88%) display low basal expression (in the lower 10 percentile of transcripts). In young leaves subjected to biotic stress, we found up-regulation of 266 of the NLRs To illustrate the utility of our tool for the positional cloning of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease-resistant varieties.

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor.

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.

Relationship between anterior mandibular bone thickness and the angulation of incisors and canines-a CBCT study.

OBJECTIVES: The morphology of the maxillary and mandibular alveolar cortex plays an important role in the planning of orthodontic treatment. Cone-beam computed tomography (CBCT) provides a precise demonstration of anatomical structures. Therefore, the aim of this paper was to evaluate what influence the position of incisors and canines have on the dimensions of the cortical and spongious bone of the anterior mandibular alveolar process. MATERIALS AND METHODS: The material consisted of 100 CBCT volumes (61 females and 39 males, aged 18-71 years) obtained by means of a Gendex GXCB-500 machine and analysed using i-CAT Vision and CorelDRAW 9 software. Several linear and angular measurements were taken of cortical and spongious mandibular, vestibular and lingual alveolar bone. RESULTS: The thickness of the vestibular spongious bone increased around lateral incisors and canines together with dental axis inclination, as did the thickness of the lingual spongious bone around central incisors and canines with greater angles of vestibular cortex curvature. In all teeth, the thickness of lingual cancellous bone decreased along with increase of the angle of tooth inclination. In the case of almost all groups of teeth, the thickness of lingual cancellous bone around teeth declined as the angle of curvature of the cortical bone decreased. The rotation of mandibular incisors and canines did not affect the thickness of the surrounding bone. CONCLUSIONS: The position of teeth has little influence on vestibular bone thickness and is only significant around central incisors. In the case of almost all groups of teeth, the thickness of lingual spongious bone around teeth declined as the angle of curvature of the cortical bone decreased. CLINICAL RELEVANCE: CBCT is a diagnostic tool that provides detailed information on the dimensions of the anterior dentate mandibular alveolar process.

Modeling of Dolichol Mass Spectra Isotopic Envelopes as a Tool to Monitor Isoprenoid Biosynthesis.

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.

Clinical and Histological Evaluation of Direct Pulp Capping on Human Pulp Tissue Using a Dentin Adhesive System.

Objective. This study presents a clinical and histological evaluation of human pulp tissue responses after direct capping using a new dentin adhesive system. Methods. Twenty-eight caries-free third molar teeth scheduled for extraction were evaluated. The pulps of 22 teeth were mechanically exposed and randomly assigned to 1 of 2 groups: Single Bond Universal or calcium hydroxide. Another group of 6 teeth acted as the intact control group. The periapical response was assayed, and a clinical examination was performed. The teeth were extracted after 6 weeks, and a histological analysis was performed. The pulp status was assessed, and the thickness of the dentin bridge was measured and categorized using a histological scoring system. Results. The clinical phase was asymptomatic for Single Bond Universal patients. Patients in the calcium hydroxide group reported mild symptoms of pain, although the histological examination revealed that dentin bridges with or without limited pulpitis had begun forming in each tooth. The universal adhesive system exhibited nonsignificantly increased histological signs of pulpitis (P > 0.05) and a significantly weaker thin mineralized tissue layer (P < 0.001) compared with the calcium hydroxide group. Conclusion. The results suggest that Single Bond Universal is inappropriate for human pulp capping; however, further long-term studies are needed to determine the biocompatibility of this agent.

Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing.

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq) with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.

Species-Specific Identification of Human Adenoviruses in Sewage.

Human adenovirus (HAdV) diversity in sewage was assessed by species-specific molecular methods. Samples of raw sewage were collected in 14 sewage disposal systems from January to December 2011, in Poland. HAdVs were detected in 92.1% of the analysed sewage samples and was significantly higher at cities of over 100 000 inhabitants. HAdV DNA was detected in sewage during all seasons. The most abundant species identified were HAdV-F (average 89.6%) and -A (average 19.6%), which are associated with intestine infections. Adenoviruses from B species were not detected. The result of the present study demonstrate that human adenoviruses are consistently present in sewage in Poland, demonstrating the importance of an adequate treatment before the disposal in the environment. Multiple HAdV species identified in raw sewage provide new information about HAdV circulation in the Polish population.

Environmental Surveillance of Non-polio Enteroviruses in Poland, 2011.

The aim of this study was to apply environmental surveillance to evaluate circulation of non-polio enteroviruses (NPEVs) in sewage in Poland. Samples of raw sewage were collected in 14 sewage disposal systems from January to December, 2011. Sewage samples were concentrated prior to analysis by RT-PCR and isolation in cells (RD, L20B and Caco-2). Out of the 165 analysed samples, 127 (77%) were positive for enteroviruses using RT-PCR and 109 (66%) were positive for enteroviruses using cell culture methods and the highest detection rate was observed in the summer and autumn. In total, 141 enteroviruses were identified using neutralization test (107 NPEVs and 34 polioviruses). Accounting for 52% of all the detected NPEVs, E11 and E3 were the predominant serotypes identified in raw sewage. Retrospectively, E11 was the known aetiology for the past aseptic meningitis outbreaks in Poland, as E3 being rarely associated with any outbreak prior to 2013. In conclusion, the environmental surveillance provides data which may help in understanding the epidemiology of enteroviruses in humans.

The utility of Caco-2 cells in isolation of enteroviruses from environmental and clinical material.

The work presented here demonstrates the utility of Caco-2 cells in the isolation of enteroviruses (EVs) from environmental and clinical materials. Thirty-two samples of cerebrospinal fluid positive in Pan-entero RT-PCR were taken for EV strain isolation in cell culture. Out of the 32 samples analysed, 22 (68.75%) were positive for enteroviruses by isolation in Caco-2 cells, and 10 (31.25%) were positive by isolation in RD cells. High viral titre in clinical specimens resulted in rate increase for isolation in Caco-2 cells and RD cells (87.5% and 50%, respectively). Also, the probability of isolation of enteroviruses from sewage in Caco-2 cells was 20 times higher that in RD cells. We proved that Caco-2 cells were more effective than RD cells in enterovirus isolation, irrespective of the material used in the inoculation process.

The detection of enteroviruses in sewage using Caco-2 cells.

The work presented here demonstrates the utility of Caco-2 cells to detect enteroviruses in sewage. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by cell culture assays and RT-PCR. Enteroviruses were detected in all sewage samples, but only one sample was positive solely in RT-PCR assay. We proved that Caco-2 cells were more effective than RD and L20B cells in enterovirus isolation, depending on procedures used in the inoculation process.

[Identification of infection agent in diagnostic material from patients with suspicion of enteroviral meningitis]. / Identyfikacja czynnika zakaznego w materiale diagnostycznym od chorych z podejrzeniem enterowirusowego zapalenia osrodkowego ukladu nerwowego.

Clinical diagnosis of enteroviral infections of the central nervous system are performed by virus isolation in sensitive cell lines and RT-PCR assay. The aim of the study was evaluation these techniques for fast diagnosis meningitis caused by enteroviruses. 69 samples (cerebrospinal fluid, CSF) were collected and analised by RT-PCR reaction. 39 samples were positive (56.5%). 20 positive sample were selected and simultaneously 20 stool samples from the same patients were collected for virus isolation in sensitive cell line. Positive isolation was observed only in one CSF (5.3%) and in 9 stool samples (45%).

[Enteroviruses serotypes isolated in years 2008-2009 in NIPH-NIH]. / Serotypy enterowirusów wyizolowanych w latach 2008-2009 w zakladzie wirusologii NIZP - PZH.

Enteroviruses are important etiologic agents of many human diseases such as diarrhea, self-limiting gastroenteritis, respiratory infections, conjunctivitis, hepatitis, aseptic meningitis, encephalitis, and paralysis. The aim of this study was the evaluation of the frequency of enteroviral infections in Poland in 2008-2009. Out of 178 clinical materials tested for the presence of enteroviruses, 24 samples (13,5%) were positive. In the case of 153 samples from patients suffering from acute flaccid paralysis (AFP), positive results were obtained for 6 samples (4%). Moreover, 25 samples coming from patients with clinical symptoms caused by nonpoliomyelitic enteroviruses were analyzed, giving 18 positive results (72%). The most frequently isolated enterovirus serotypes were Coxsackie B (25%), ECHO30 (25%) i ECHO6 (21%).

[Virological and molecular methods in enteroviruses infections in 2004-2008, Poland]. / Badania wirusologiczne i molekularne w zakazeniach enterowirusowych w Polsce w latach 2004-2008.

The availability of sensitive cell lines and molecular methods have allowed for epidemiological surveillance of nonpoliomyelitic enteroviruses in Poland in 2004-2008. The aim of the study was evaluation ofenterovirus diagnosis based on RT-PCR reaction. Available data collected in 2004-2008 indicated that 116 isolation of enteroviruses were performed. The most frequently isolated enterovirus serotype was Coxsackievirus B (30.17%). Other enteroviruses serotypes isolated during study period were Coxsackievirus A (8.62%), ECHO3 (1.72%), ECHO6 (7.76%), ECHO7 (4.31%), ECHO25 (0.86%), ECHO30 (18.97%), poliovirus (0.86%).

Conserved Cys residue influences catalytic properties of potato endo-(1-->3)-beta-glucanase GLUB20-2.

The synthesis and degradation of (1-->3)-beta-glycosidic bonds between glucose moieties are essential metabolic processes in plant cell architecture and function. We have found that a unique, conserved cysteine residue, positioned outside the catalytic centre of potato endo-(1-->3)-beta-glucanase - product of the gluB20-2 gene, participates in determining the substrate specificity of the enzyme. The same residue is largely responsible for endo-(1-->3)-beta-glucanase inhibition by mercury ions. Our results confirm that the spatial adjustment between an enzyme and its substrate is one of the essential factors contributing to the specificity and accuracy of enzymatic reactions.