Sensor histidine kinases (SHKs) are an integral component of the molecular machinery that permits bacteria to adapt to widely changing environmental conditions. CpxA, an extensively studied SHK, is a multidomain homodimeric protein with each subunit consisting of a periplasmic sensor domain, a transmembrane domain, a signal-transducing HAMP domain, a dimerization and histidine phospho-acceptor sub-domain (DHp) and a catalytic and ATP-binding subdomain (CA). The key activation event involves the rearrangement of the HAMP-DHp helical core and translation of the CA towards the acceptor histidine, which presumably results in an autokinase-competent complex. In the present work we integrate coarse-grained, all-atom, and hybrid QM-MM computer simulations to probe the large-scale conformational reorganization that takes place from the inactive to the autokinase-competent state (conformational step), and evaluate its relation to the autokinase reaction itself (chemical step). Our results highlight a tight coupling between conformational and chemical steps, underscoring the advantage of CA walking along the DHp core, to favor a reactive tautomeric state of the phospho-acceptor histidine. The results not only represent an example of multiscale modelling, but also show how protein dynamics can promote catalysis.

The histidine kinases belong to the family of two-component systems, which serves in bacteria to couple environmental stimuli to adaptive responses. Most of the histidine kinases are homodimers, in which the HAMP and DHp domains assemble into an elongated helical region flanked by two CA domains. Recently, X-ray crystallographic structures of the cytoplasmic region of the Escherichia coli histidine kinase CpxA were determined and a phosphotransferase-defective mutant, M228V, located in HAMP, was identified. In the present study, we recorded 1 μs molecular dynamics trajectories to compare the behavior of the WT and M228V protein dimers. The M228V modification locally induces the appearance of larger voids within HAMP as well as a perturbation of the number of voids within DHp, thus destabilizing the HAMP and DHp hydrophobic packing. In addition, a disruption of the stacking interaction between F403 located in the lid of the CA domain involved in the auto-phosphorylation and R296 located in the interacting DHp region, is more often observed in the presence of the M228V modification. Experimental modifications R296A and R296D of CpxA have been observed to reduce also the CpxA activity. These observations agree with the destabilization of the R296/F403 stacking, and could be the sign of the transmission of a conformational event taking place in HAMP to the auto-phosphorylation site of histidine kinase. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 670-682, 2016.

Hereditary hemochromatosis is an autosomal recessive disorder characterized by severe iron overload. It is usually associated with homozygosity for the HFE gene mutation c.845G>> A; p.C282Y. However, in some cases, another HFE mutation (c.187C>> G; p.H63D) seems to be associated with the disease. Its penetrance is very low, suggesting the possibility of other iron genetic modulators being involved. In this work, we have screened for HAMP promoter polymorphisms in 409 individuals presenting normal or increased serum ferritin levels together with normal or H63D-mutated HFE genotypes. Our results show that the hepcidin gene promoter TG haplotype, originated by linkage of the nc.-1010C>> T and nc.-582A>> G polymorphisms, is more frequent in the HFE_H63D individuals presenting serum ferritin levels higher than 300 μg/L than in those presenting the HFE_H63D mutation but with normal serum ferritin levels or in the normal control group.Moreover, it was observed that the TG haplotype was associated to increased serum ferritin levels in the overall pool of HFE_H63D individuals. Thus, our data suggest that screening for these polymorphisms could be of interest in order to explain the phenotype. However, this genetic condition seems to have no clinical significance.

Does the hormone of iron metabolism, hepcidin, exhibit 'self-control'? Hepcidin is a small, disulfide-rich peptide synthesized by the liver, which plays a keystone role in regulating systemic iron metabolism in mammals. Hepcidin acts by binding and triggering the lysosomal degradation of the cellular iron exporter ferroportin. Ultimately, decreased ferroportin leads to decreased plasma iron levels. Although various modulators of HAMP (the hepcidin antimicrobial peptide gene) expression are known, no auto-regulatory pathway has been described. In their paper published in the Biochemical Journal in April 2013, Pandur et al. identify an auto-regulatory pathway in which prohepcidin regulates HAMP expression. The authors observe that prohepcidin can bind to the inflammation-regulated STAT3 (signal transducer and activator of transcription 3)-binding site in the HAMP promoter to negatively regulate HAMP expression. Furthermore, the authors find that the prohepcidin-binding partner, α-1 antitrypsin, inhibits prohepcidin's ability to decrease HAMP activity. This is significant as α-1 antitrypsin, similar to hepcidin, is an acute-phase reactant that is up-regulated by inflammation. In conclusion, the discovery of a hepcidin auto-regulatory pathway, first, supports the emerging notion that hepcidin regulation is exquisitely fine-tuned through a process of combinatorial control; and secondly, suggests that hepcidin may play a hand in its own deregulation in diseases of iron metabolism that involve aberrant cytokine signalling (e.g. the anaemia of inflammation).

OBJECTIVE

Despite the obligate iron loss from blood donation, some donors present with hyperferritinaemia that can result from a wide range of acute and chronic conditions including hereditary haemochromatosis (HH). The objective of our study was to investigate the causes of hyperferritinaemia in the blood donor population and explore the value of extensive HH mutational analyses.

METHODS

Forty-nine consecutive donors (f = 6, m = 43) were included prospectively from the Capital Regional Blood Center. Inclusion criteria were a single ferritin value >1000 μg/l or repeated hyperferritinaemia with at least one value >500 μg/l. All donors were questioned about their medical history and underwent a physical examination, biochemical investigations and next-generation sequencing of HH-related genes, including the HFE gene, the haemojuvelin gene (HFE2/HJV), the hepcidin gene (HAMP), the ferroportin 1 gene (SLC40A1) and the transferrin receptor 2 gene (TFR2).

RESULTS

Forty of 49 donors were mutation positive with a combined 69 mutations, 54 of which were located in the HFE gene. There were 11 mutations in the TFR2 gene, two mutations in the HFE2 gene and two mutations in the HAMP gene. Only four donors had apparent alternative causes of hyperferritinaemia.

CONCLUSIONS

HH-related mutations were the most frequent cause of hyperferritinaemia in a Danish blood donor population, and it appears that several different HH-genotypes can contribute to hyperferritinaemia. HH screening in blood donors with high ferritin levels could be warranted. HH-related iron overload should not in itself result in donor ineligibility.

The discovery of the HFE, HJV, HAMP, TfR2, and SLC40A1 genes and preliminary understanding of their roles in iron homeostasis have contributed tremendously to our understanding of the pathogenesis of genetic hemochromatosis. Although several new models of iron metabolism have been proposed, some key "sensor" steps of iron absorption in the enterocytes and of iron storage in hepatocytes and other cells remain unclear. A diagnosis of non-HFE genetic hemochromatosis should be considered in patients with unexplained iron overload who do not have the common mutations in the HFE genes. Phenotypic evaluation such as liver biopsy and measurement of hepatic iron concentration remain important in non-HFE hemochromatosis because mutations in other genes are rare and there are no other available noninvasive tests to confirm the diagnosis. Phlebotomy remains the mainstay of therapy also for non-HFE hemochromatosis. However, phlebotomy may not be well tolerated in certain forms of non-HFE hemochromatosis such as "ferroportin disease."

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells.

Patients with hereditary hemochromatosis (HC) may present a plenty of clinical symptoms, thus are referred to various specialists and may prone a significant diagnostic dillema. The molecular basis of hemochromatosis is more complex than expected. In 1996 HFE gene was identified and its main mutations (C282Y and H63D) were described as well as their high frequency in population of European descent. Them: Most patients with clinical symptoms of hemochromatosis are homozygous for C282Y but it is also clear that some families are linked to rarer conditions, named "non-HFE hemochromatosis". Between 2000-2004 other genes involved in iron homeostasis were intensively studied, leading to recognition of hepcidin (HAMP) - the most important iron hormone, hemojuvelin (HJV), transferin receptor 2 (TfR2) and ferroportin. Recent findings led to novel hypothesis on potential digenic modes of inheritance or the involvement of modifier genes. Hepcidin plays a central role in mobilization of iron, HFE, TfR2 and HJV playing a modulating role in its production, related to the body's iron status. It has also been demonstrated that HAMP negatively regulates cellular iron efflux by affecting the ferroportin cell surface availability. The result of such a wide investigations is OMIM classification of hereditaty hemochromatosis, typing four types of the disease.

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. In this study, we firstly revealed that a new alternative HAMP transcript was found in hepatoma-derived cell line HLF, which was identical to the wild-type preprohepcidin sequence except lacking of an internal 60 bases. In addition to HLF, most of hepatoma-derived cell lines have significant copy numbers of variant-type hepcidin mRNA by a copy-based-digital PCR. Furthermore, the copy number of hepcidin mRNA variant was significantly higher in serum exosomes of hepatocellular carcinoma patients. The quantification of exosomal hepcidin mRNA variant may serve as a potential new biomarker for HCC diagnosis.

OBJECTIVE

Recent discoveries in molecular mechanisms of iron metabolism have changed the classical view of hereditary iron overload conditions. We present natural mutations in newly discovered genes and related phenotypes observed in patients with different form of haemochromatosis.

BACKGROUND

Most haemochromatosis patients are homozygous for the C282Y mutation in the HFE gene. Ferroportin, TFR2, hemojuvelin and hepcidin mutations also cause iron overload. Recent data support the hypothesis that haemochromatosis should no longer be considered a monogenic disease but rather an oligogenic disorder. Several results suggest that haemochromatosis could result from digenic inheritance of mutations in HFE and HAMP.

CONCLUSIONS

Other modifier genes probably influence penetrance in C282Y homozygous patients. Such genes could enhance or reduce the phenotypic expression in various iron overload conditions.

BACKGROUND

There are significant changes in the abbreviated injury scale (AIS) 2005 system, which make it impractical to compare patients coded in AIS version 98 with patients coded in AIS version 2005.

METHODS

Harborview Medical Center created a computer algorithm "Harborview AIS Mapping Program (HAMP)" to automatically convert AIS 2005 to AIS 98 injury codes. The mapping was validated using 6 months of double-coded patient injury records from a Level I Trauma Center. HAMP was used to determine how closely individual AIS and injury severity scores (ISS) were converted from AIS 2005 to AIS 98 versions. The kappa statistic was used to measure the agreement between manually determined codes and HAMP-derived codes.

RESULTS

Seven hundred forty-nine patient records were used for validation. For the conversion of AIS codes, the measure of agreement between HAMP and manually determined codes was [kappa] = 0.84 (95% confidence interval, 0.82-0.86). The algorithm errors were smaller in magnitude than the manually determined coding errors. For the conversion of ISS, the agreement between HAMP versus manually determined ISS was [kappa] = 0.81 (95% confidence interval, 0.78-0.84).

CONCLUSIONS

The HAMP algorithm successfully converted injuries coded in AIS 2005 to AIS 98. This algorithm will be useful when comparing trauma patient clinical data across populations coded in different versions, especially for longitudinal studies.

Hereditary iron overload is mainly due to mutations of the HFE gene, implicated in most cases of hereditary hemochromatosis. Non-HFE-related hereditary iron overload is rare. It includes hereditary hemochromatosis related to mutations of other genes, ferroportin disease (also known as hemochromatosis type 4), and entities associated with specific clinical manifestations. Four genes have been implicated in hereditary hemochromatosis: HFE and TFR2 (which codes for the second transferrin receptor), both involved in adult forms of hereditary hemochromatosis, and HAMP and HJV, which code for hepcidin and hemojuvelin, respectively, and are responsible for juvenile hemochromatosis. All types of hereditary hemochromatosis share common clinical and biological characteristics, including an autosomal recessive inheritance pattern, elevation of transferrin saturation as the initial manifestation, hepatic parenchymal iron overload, and sensitivity to therapeutic phlebotomy. They are due to hyperabsorption of dietary iron and are linked to a deficit of hepcidin, the principal iron regulator in the body. Ferroportin disease is a special dominantly inherited clinical form of iron overload due to mutations of the SLC40A1 gene. Its expression differs significantly from that of hereditary hemochromatosis, and its mechanism is related to impairment of iron release from reticuloendothelial cells. Other causes of non-HFE-related hereditary iron overload are usually associated with recognizable clinical manifestations, such as anemia or neurological disorders.

The present study aimed to observe the identification of biomarkers of silicosis based on the differentially expressed serum proteins between normal healthy individuals and patients with silicosis fibrosis. A total number of 20 patients with clinically diagnosed silicosis were screened, which were designated as the foundation treatment group. In addition, 20 age-matched healthy patients attending a check-up at the physical examination department were selected. Serum samples were obtained and a combined protein chip with surface-enhanced laser desorption ionization flight mass spectrometry was applied to perform serum analysis. Data preprocessing, screening differences in peak, hierarchical cluster analysis, Principal Component Analysis, construction of a decision tree model, and prediction based on the differences between peaks corresponding to proteins were performed to analyze the data. The results revealed differences in the proteins in serum between the normal group and the group prior to foundation treatment prediction. The corresponding names of the protein peak, predicted protein, and gene name were as follows: M1948_00, complement c3 frag, C3; M2017_02, amyloid-βa4 protein, APP; and M2879_56, hepcidin, HAMP. Differentially expressed serum proteins in the normal group and the basis treatment group were predicted, including M2017_02, amyloid-βa4 protein, APP; M2879_56, hepcidin, HAMP; and M3224_97, fibrinogen-α chain frags, FGA. The differentially expressed serum proteins in the group prior to basis treatment and the group following basis treatment were predicted, including M2001_69, amyloid-βa4 protein, APP; M2017_02, amyloid-βa4 protein, APP, M4144_81, plasma protease c1 inhibitor frag, and SERPING1. In conclusion, there were differences in the proteins in serum between the patients with silicosis fibrosis and healthy individuals.

Tebuconazole (TEB) is one of the widely used broad-spectrum triazole fungicides. Its accumulation in mammals leads to various endocrine disruptions. However, it is unclear whether the exposure of TEB during pregnancy affects the growth and development of fetus and placenta. Here, TEB was exposed to pregnant Sprague-Dawley female rats from gestational days 12-21 of 0, 25, 50 or 100 mg/kg for 10 days. TEB reduced placental estradiol levels. TEB disrupted the structure and function of the placenta, leading to hypertrophy, fibrin exudation, edema, calcification, arterial fibroblast proliferation, and trophoblastic infarction. RNA-seq analysis showed that TEB mainly down-regulated the expression of iron transport genes and up-regulated the expression of genes for immune/inflammatory responses. Further qPCR showed that TEB down-regulated Tfrc, Hamp, Eif2ak2 and up-regulated the expression of Cd34, Cd36, Jag1, Pln, Cyp1a1, Esrra, and Aqp1 at 50 and 100 mg/kg. Western blot and semi-quantitative immunohistochemical staining also demonstrated that TEB lowered the levels of TFRC and EIF2AK2 and increased the levels of CD34, CD36, JAG1, CYP1A1, and ESRRA at 50 and 100 mg/kg. In conclusion, TEB severely damages the structure and function of the placenta, leading to hypertrophy of the placenta, low birth weight and feminization of the male fetus possibly via several pathways including iron transport and TNF signaling.

Keywords: Estradiol; Fertilization; Low birth weight; Placental dysfunction; Tebuconazole; Trophoblast.

Medical Physics is the scientific healthcare profession concerned with the application of the concepts and methods of physics in medicine. The European Federation of Organisations for Medical Physics (EFOMP) acts as the umbrella organization for European Medical Physics societies. Due to the rapid advancements in related scientific fields, medical physicists must have continuous education through workshops, training courses, conferences, and congresses during their professional life. The latest developments related to this increasingly significant medical speciality were presented during the 1st European Congress of Medical Physics 2016, held in Athens, September 1-4, 2016, organized by EFOMP, hosted by the Hellenic Association of Medical Physicists (HAMP), and summarized in the current volume.

According to the European Federation of Organizations for Medical Physics (EFOMP) policy statement No. 13, "The rapid advance in the use of highly sophisticated equipment and procedures in the medical field increasingly depends on information and communication technology. In spite of the fact that the safety and quality of such technology is vigorously tested before it is placed on the market, it often turns out that the safety and quality is not sufficient when used under hospital working conditions. To improve safety and quality for patient and users, additional safeguards and related monitoring, as well as measures to enhance quality, are required. Furthermore a large number of accidents and incidents happen every year in hospitals and as a consequence a number of patients die or are injured. Medical Physicists are well positioned to contribute towards preventing these kinds of events". The newest developments related to this increasingly important medical speciality were presented during the 8th European Conference of Medical Physics 2014 which was held in Athens, 11-13 September 2014 and hosted by the Hellenic Association of Medical Physicists (HAMP) in collaboration with the EFOMP and are summarized in this issue.

The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivo conditions, maintaining the intracellular milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilized Escherichia coli cells. In this method, E. coli expressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a large sulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide cross-linking to interact.

Miller LD, Coffman LG, Chou JW et al. An iron regulatory gene signature predicts outcome in breast cancer. Cancer Res. 71(21), 6728-6737 (2011). In breast cancer, recent progress in technology has enabled us to define different prognostic genetic signatures. Based upon them, breast tumors have been grouped into the four principal categories: basal-like or triple-negative, erbB2-positive, normal-like, and luminal type (A and B); with luminal types sharing the expression of estrogen receptor- and/or progesterone receptor-related genes and, basal-like and erbB2-positive subgroups associated with worse prognosis. So far, Oncotype DX(®) (Genomic Health Inc., Redwood City, CA, USA), Mammaprint(®) (Agendia Inc, Huntington Beach, CA, USA), the Breast Cancer Index(®) (BCI, Biotheranostics, San Diego, CA, USA) and PAM50 (Expression Analysis Inc., Durham, NC, USA) are the only multigene assays that have been marketed in North America and Europe. However, any genetic signature assay still has to gain acceptance as a validated assay before introduction into current clinical practice. This study describes an iron regulatory gene signature (IRGS) in breast cancer associated with clinical outcome. Within the molecular luminal type, the IRGS provides prognostic information similar to Oncotype DX and gene sets selected to assess proliferation. In spite of this, it is relevant that two complementary pathways that are regulatory of iron metabolism - the iron export (Fp/HAMP) and the iron import (TFRC/HFE) gene dyads - were embedded in the IRGS gene set and were associated with clinical outcome as well. Differences in metabolic pathways between cancer and normal cells have been widely described, and potential applications for more refined therapy have been proposed by expanding genetic signature assessment technology to concomitant metabolic pathways investigation. Consistent with this, it is reasonable to imagine that the iron-export and the iron-import gene dyads will be considered potential targets for treatment of breast cancer patients expressing the IRGS genes.

The hormone hepcidin plays a central role in controlling iron homeostasis. Iron-mediated hepcidin synthesis is triggered via the BMP/SMAD pathway. At inflammation, mainly IL-6 pro-inflammatory cytokine mediates the regulation of hepcidin via the JAK/STAT signalling pathway. Microglial cells of the central nervous system are able to recognize a broad spectrum of pathogens via toll-like receptors and initiate inflammatory response. Although the regulation of hepcidin synthesis is well described in many tissues, little is known about the inflammation mediated hepcidin regulation in microglia. In this study, we investigated the pathways, which are involved in HAMP regulation in BV2 microglia due to inflammatory mediators and the possible relationships between the iron regulatory pathways. Our results showed that IL-6 produced by resting BV2 cells was crucial in maintaining the basal HAMP expression and hepcidin secretion. It was revealed that IL-6 neutralization decreased both STAT3 and SMAD1/5/9 phosphorylation suggesting that IL-6 proinflammatory cytokine is necessary to maintain SMAD1/5/9 activation. We revealed that IL-6 influences BMP6 and TMPRSS6 protein levels, moreover it modified TfR2 expression, as well. In this study, we revealed that BV2 microglia increased their hepcidin secretion upon IL-6 neutralization although the major regulatory pathways were inhibited. Based on our results it seems that both at inflammation and at normal condition the absence of IL-6 triggered HAMP transcription and hepcidin secretion via the NFκB pathway and possibly by the autocrine effect of TNFα cytokine on BV2 microglia.

Keywords: BMP/SMAD; Hepcidin; Inflammation; Iron metabolism; Microglia; STAT3.

Despite the crucial role of the liver as the central regulator of iron homeostasis, no studies have directly tested the modulation of liver gene and protein expression patterns during iron deficiency instauration and recovery with fermented milks. Fermented goat milk consumption improves the key proteins of intestinal iron metabolism during iron deficiency recovery, enhancing the digestive and metabolic utilization of iron. The aim of this study was to assess the influence of fermented goat or cow milk consumption on liver iron homeostasis during iron-deficiency anemia recovery with normal or iron-overload diets. Analysis included iron status biomarkers, gene and protein expression in hepatocytes. In general, fermented goat milk consumption either with normal or high iron content up-regulated liver DMT1, FPN1 and FTL1 gene expression and DMT1 and FPN1 protein expression. However, HAMP mRNA expression was lower in all groups of animals fed fermented goat milk. Additionally, hepcidin protein expression decreased in control and anemic animals fed fermented goat milk with normal iron content. In conclusion, fermented goat milk potentiates the up-regulation of key genes coding for proteins involved in iron metabolism, such as DMT1, and FPN1, FTL1 and down-regulation of HAMP, playing a key role in enhanced iron repletion during anemia recovery, inducing a physiological adaptation of the liver key genes and proteins coordinated with the fluctuation of the cellular iron levels, favoring whole-body iron homeostasis.

The complexation of molybdate with the nucleotides adenosine-5'-monophosphate (5'-AMP), adenosine-3'-monophosphate (3'-AMP) and guanosine-5'-monophosphate (5'-GMP) has been investigated by (1)H and (31)P NMR and Mo K-edge X-ray absorption near edge (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy. Acidification of aqueous solutions containing molybdate and each of the nucleotides resulted in the formation of a single species characterized by (1)H resonances which are deshielded relative to those of free nucleotide. Analysis of the two-component systems indicated a Mo/nucleotide ratio of 2.5:1 for the complexation species. White compounds, characterized as Na(2)[Mo(5)O(15)(HB)(2)] (B=5'-AMP, 5'-GMP), have been isolated from the acidified molybdate/H(2)B solutions. Dissolution in D(2)O replicates the NMR spectra of the solution species observed prior to precipitation. Solution and solid state Mo K-edge XAS and EXAFS spectroscopy of Na(2)[Mo(5)O(15)(HAMP)(2)] and Na(6)[Mo(5)O(15)(PO(4))(2)] provide convincing evidence for the presence of a pentamolybdodiphosphate core in the molybdate-nucleotide complexes in both the solid and solution states.

Plants have a perception system triggered by pathogen and pest signals to initiate defense. These signals include evolutionarily conserved molecules from microbes and insects termed pathogen/herbivore-associated molecular patterns (PAMPs/HAMPs). Here we showed that hexaacetyl-chitohexaose (HC), an oligosaccharide from chitin, a structural component in insect exoskeletons and fungi cell walls, upregulated defense-associated genes WRKY22, GST1, RAR1, EDS1, PAL1 and NPR2, and downregulated ICS1 at 1 h after HC treatment in Sun Chu Sha mandarin leaves. The effect was transient as defense gene transcriptional changes were not observed at 18 h after the treatment. Electrical penetration graph (EPG) recordings were used to study the feeding behavior of Asian citrus psyllid (ACP) following the HC treatment. ACP is the hemipteran vector of Candidatus Liberibacter asiaticus (CLas), the pathogen associated with huanglongbing (HLB). Adult ACP displayed reduced intercellular probing, reduced xylem feeding count and duration, and increased non-probing activity on HC-treated citrus compared to controls. During an 18-h recording, percentage for total duration of xylem ingestion, phloem ingestion, intercellular probing were lower, and the percentage of non-probing behavior was higher in HC-treated leaves than in controls. In host-selection behavior studies, HC treatment did not alter the attractiveness of citrus leaves under light or dark conditions. In addition, ACP feeding on HC-treated leaves did not show differences in mortality for up to 10 day of exposure. In summary, we report that HC induced a transient defense in citrus and an inhibitory effect on ACP feeding but did not affect host selection or the insect fitness under the tested conditions.

OBJECTIVE

During the last years hyperthermia is a developing therapeutic modality in Greece. Quality assurance (QA) procedures are essential for ensuring the correct operation of the hyperthermia system and therefore the selective heating of the tumor with minimum toxicity to the surrounding healthy tissues. The European Society for Hyperthermic Oncology (ESHO) has proposed QA guidelines for superficial as well as deep hyperthermia systems. The purpose of this study was to describe the adapted QA protocol for superficial and deep hyperthermia systems established in Greece.

METHODS

A working group was created by the Hellenic Association of Medical Physicists (HAMP) for the proposal of QA guidelines for superficial and deep hyperthermia systems. A review of the protocol proposed by ESHO, together with the existing protocols in other European and International centers, as well as protocols suggested by European or International organizations, was performed. Then, a protocol was suggested, describing procedures for QA according to the current technology and the existing equipment used in Greece.

RESULTS

A protocol describing the procedures for QA of superficial and deep hyperthermia systems was proposed. These procedures aim to evaluate the correct operation of the device, the thermometric system, the generator, the incorporated power meter and the applicators. It will also ensure the electrical safety of the devices.

CONCLUSIONS

The proposed protocol, applied by medical physicists in Greece, will ensure an efficient treatment with safety and minimum adverse effects. This protocol has been approved by the Hellenic Society of Oncologic Hyperthermia and the Hellenic Association of Medical Physicists.

Although distinct amino acid motifs containing consecutive prolines (polyP) cause ribosome stalling, which necessitates recruitment of the translation elongation factor P (EF-P), they occur strikingly often in bacterial proteomes. For example, polyP motifs are found in more than half of all histidine kinases in Escherichia coli K-12, which raises the question of their role(s) in receptor function. Here we have investigated the roles of two polyP motifs in the osmosensor and histidine kinase EnvZ. We show that the IPPPL motif in the HAMP domain is required for dimerization of EnvZ. Moreover, replacement of the prolines in this motif by alanines disables the receptor's sensor function. The second motif, VVPPA, which is located in the periplasmic domain, was found to be required for interaction with the modulator protein MzrA. Our study also reveals that polyP-dependent stalling has little effect on EnvZ levels. Hence, both polyP motifs in EnvZ are primarily involved in protein-protein interaction. Furthermore, while the first motif occurs in almost all EnvZ homologues, the second motif is only found in species that have MzrA, indicating co-evolution of the two proteins.