Germline mutations of the RET proto-oncogene (RET), its ligand glial cell-derived neurotrophic factor (GDNF), and neurturin (NTN) gene have been reported in patients with Hirschsprung's disease. A targeted mutation in the tyrosine kinase domain of RET produced total intestinal aganglionosis and renal agenesis in homozygous transgenic mice. Here we describe a homozygous mutation of the human gene for the RET tyrosine kinase domain that was present in a male neonate with total intestinal aganglionosis. Gut wall biopsy specimens from the stomach to the anorectum showed no ganglion cells. No urinary tract abnormalities were detected. Genomic DNAs were isolated from peripheral blood lymphocytes of the infant and his parents. DNA sequences of all the RET/GDNF/NTN coding regions were determined using a direct DyeDeoxy Terminator Cycle method. A homozygous missense mutation (CGG-to-TGG) at RET codon 969 was identified in this patient, which resulted in an amino acid change from arginine to tryptophan. No germline RET/GDNF/NTN mutations were found in his parents. In this case, the homozygous RET mutation seemed to cause a critical alteration of the Ret tyrosine kinase activity, which resulted in total intestinal aganglionosis but not renal agenesis. Discrepancies in phenotypic expression between humans and mice suggest differing threshold values for RET signal transduction in species or organs.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFR alpha-1 and GFR alpha-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFR alpha-1 and GFR alpha transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFR alpha-2 mRNA after 24 hr of treatment with 10(-10) M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFR alpha-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFR alpha-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFR alpha-1 and GFR alpha-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma.

N-Acylhomoserine lactone (AHL)-acylase (also known as amidase or amidohydrolase) is a class of enzyme that belongs to the Ntn-hydrolase superfamily. As the name implies, AHL-acylases are capable of hydrolysing AHLs, the most studied signaling molecules for quorum sensing in Gram-negative bacteria. Enzymatic degradation of AHLs can be beneficial in attenuating bacterial virulence, which can be exploited as a novel approach to fight infection of human pathogens, phytopathogens or aquaculture-related contaminations. Numerous acylases from both prokaryotic and eukaryotic sources have been characterized and tested for the interference of quorum sensing-regulated functions. The existence of AHL-acylases in a multitude of organisms from various ecological niches, raises the question of what the physiological roles of AHL-acylases actually are. In this review, we attempt to bring together recent studies to extend our understanding of the biological functions of these enzymes in nature.

Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.

The coat protein (CP) gene of 75 South African Potato virus Y (PVY) isolates was amplified using reverse-transcriptase polymerase chain reaction (RT-PCR). The resulting cDNA products were cloned and sequenced. These sequences were used to identify the strains to which the isolates belonged. Some, when compared to reference sequences, belonged to the PVY(N) and PVY(O) strains. A number of isolates were found to demonstrate significant homology to PVY(N) strains from China. A large number of South African isolates possessed CP sequences showing evidence of recombination between PVY(N) and PVY(O) strains, similar to those of PVY(NTN) isolates. Multiplex RT-PCR analysis allowed further differentiation of PVY(O) isolates and revealed that the majority were of the PVY(N)-Wilga strain. It was deduced that the most likely way in which these isolates reached South Africa was via the importation of infected material.

We examined the relation between glomerular expression of chemokines from alpha-subfamily (macrophage inflammatory protein-2, MIP-2) and beta-subfamily (monocyte chemoattractant protein-1, MCP-1) and infiltration of neutrophils and monocytes in antibody mediated glomerulonephritis in rats. In the accelerated model of nephrotoxic nephritis (NTN), glomerular expression of MIP-2 and MCP-1 genes correlated with the sequential migration of neutrophil and monocyte influx, respectively. These relationships were investigated further in the heterologous phase of NTN by applying various treatments known to modulate the severity of injury. Pretreatment with bacterial lipopolysaccharide resulted in greater injury, MIP-2 expression increased 25- to 50-fold, and the glomerular neutrophil count increased two- to fourfold. Both MIP-2 mRNA levels and neutrophil infiltration were reduced by additional pretreatment with IL-6, IL-1 receptor antagonist, soluble IL-1 receptor or soluble TNF receptor (Spearman correlation coefficient r = 0.897, P < 0.005). In the heterologous phase of NTN, different pre-treatments only resulted in trivial changes in MCP-1 expression and monocyte infiltration. In conclusion, glomerular MIP-2 gene expression correlates with neutrophil infiltration both temporally during the evolution of nephritis, and when glomerular injury is modified by treatment. Glomerular MCP-1 gene expression correlates with monocyte influx. The data show chemokines of alpha- and beta-subfamilies co-operative to cause selective and sequential migration of different leukocyte subsets during development of antibody mediated glomerulonephritis.

OBJECTIVE

Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas.

METHODS

Fertile chicken eggs were incubated from E0 at 38 degrees C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression.

RESULTS

QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/ITGNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium.

CONCLUSIONS

Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression.

A vast amount of data on the natural resistance of Saccharomyces cerevisiae to a diverse array of chemicals has been generated over the past decade (chemical genetics). We endeavored to use this data to better characterize the "systems" level properties of this phenomenon. By collating data from over 30 different genome-scale studies on growth of gene deletion mutants in presence of diverse chemicals, we assembled the largest currently available gene-chemical network. We also derived a second gene-gene network that links genes with significantly overlapping chemical-genetic profiles. We analyzed properties of these networks and investigated their significance by overlaying various sources of information, such as presence of TATA boxes in their promoters (which typically correlate with transcriptional noise), association with TFIID or SAGA, and propensity to function as phenotypic capacitors. We further combined these networks with ubiquitin and protein kinase-substrate networks to understand chemical tolerance in the context of major post-translational regulatory processes. Hubs in the gene-chemical network (multidrug resistance genes) are notably enriched for phenotypic capacitors (buffers against phenotypic variation), suggesting the generality of these players in buffering mechanistically unrelated deleterious forces impinging on the cell. More strikingly, analysis of the gene-gene network derived from the gene-chemical network uncovered another set of genes that appear to function in providing chemical tolerance in a cooperative manner. These appear to be enriched in lineage-specific and rapidly diverging members that also show a corresponding tendency for SAGA-dependent regulation, evolutionary divergence and noisy expression patterns. This set represents a previously underappreciated component of the chemical response that enables cells to explore alternative survival strategies. Thus, systems robustness and evolvability are simultaneously active as general forces in tolerating environmental variation. We also recover the actual genes involved in the above-discussed network properties and predict the biochemistry of their products. Certain key components of the ubiquitin system (e.g. Rcy1, Wss1 and Ubp16), peroxisome recycling (e.g. Irs4) and phosphorylation cascades (e.g. NPR1, MCK1 and HOG) are major participants and regulators of chemical resistance. We also show that a major sub-network boosting mitochondrial protein synthesis is important for exploration of alternative survival strategies under chemical stress. Further, we find evidence that cellular exploration of survival strategies under chemical stress and secondary metabolism draw from a common pool of biochemical players (e.g. acetyltransferases and a novel NTN hydrolase).

Convection-enhanced delivery (CED) of GDNF and NTN was employed to determine the tissue clearance of these factors from the rat striatum and the response of the dopaminergic system to a single infusion. Two doses of GDNF (15 and 3 microg) and NTN (10 microg and 2 microg) were infused into the rat striatum. Animals were euthanized 3, 7, 14, 21, and 28 days post-infusion. Brains were processed for ELISA, HPLC, and immunohistochemistry (IHC). Both doses of the infused GDNF resulted in a sharp increase in striatal GDNF levels followed by a rapid decrease between day 3 and 7. Interestingly, IHC revealed GDNF in the septum and the base of the brain 14 days after GDNF administration. Dopamine (DA) turnover was significantly increased in a dose-dependent manner for more than 7 days after a single GDNF infusion. NTN persisted in the brain for at least two weeks longer than GDNF. It also had more persistent effects on DA turnover, probably due to its precipitation in the brain at neutral pH after infusion. Our data suggest that daily or continuous dosing may not be necessary for delivering growth factors into the CNS.

Neurturin (NTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family; and, while GDNF has been shown to increase dopamine (DA) release in normal animals, the ability of NTN to alter DA release has not been previously reported. The purpose of the present study was to determine if NTN could alter striatal DA release, and to compare the effects of NTN to GDNF. Male Fischer-344 rats were given a single injection of vehicle or 5 microg NTN or GDNF into the right substantia nigra. Three weeks later microdialysis experiments were conducted to assess striatal DA release. Basal extracellular levels of striatal DA were not affected by either NTN or GDNF. However, both NTN and GDNF led to increases in amphetamine-evoked overflow of DA from the ipsilateral striatum, and there was a trend for potassium-evoked overflow to be augmented. Postmortem tissue levels of DA were decreased by approximately 20% in the striatum, and increased by approximately 100% in the substantia nigra, on the ipsilateral side of the brain compared to the contralateral side following both NTN and GDNF injection. Thus, NTN, like GDNF, can augment striatal DA release, and the magnitude of the NTN effects are similar to those of GDNF.

To determine whether neurturin (NTN), a recently identified homologue of glial cell line-derived neurotrophic factor (GDNF), is able to preserve tyrosine hydroxylase immunoreactivity (TH-IR) in a rat model of Parkinson's disease, polymer encapsulated cells genetically engineered to release NTN were implanted near the substantia nigra 1 week before a unilateral medial forebrain bundle axotomy. Animals were allowed to survive for 1 week post-axotomy. Upon sacrifice, animals that received a NTN capsule had a significantly higher percentage of TH-IR (lesioned side vs non-lesioned side) than animals that had received a capsule containing non-transfected parent cells. However, in contrast to GDNF, no reduction of turning was observed upon amphetamine rotation with NTN. Nevertheless, these results suggest that NTN might have a therapeutic value for the treatment of Parkinson's disease.

Potato virus Y (PVY) is a serious potato pathogen that affects potato seed and commercial production crops. In recent decades, novel PVY strains have been described that cause necrotic symptoms on tobacco foliage and/or potato tubers. The major PVY strains that affect potato include PVY(O) and PVY(N), which have distinct serotypes that can be differentiated by immunoassay. Other economically important strain variants are derived from recombination events, including variants that cause tuber necrotic symptoms (PVY(NTN)) and PVY(O) serotypes that cause tobacco veinal necrosis (PVY(N)-W, PVY(N:O)). Although the PVY(NTN) and PVY(N)-W variants were first reported in Europe, apparently similar strains have been appearing in North America. Confirmation of the existence of these recombinant strains in North America is important, as is whether they spread from a common source or were derived by independent recombination. Whole genome sequencing can be used to positively identify strain variants and begin to address the issue of origins. Symptomology, serology, RT-PCR, and partial sequencing of the coat protein region were used to identify isolates of the PVY(NTN), PVY(N), PVY(NA-N), and PVY(N:O) for whole-genome sequencing. Sequencing confirmed the presence of PVY(NTN) and PVY(N) isolates that were >99% identical to European sequences deposited in GenBank in the 1990's. Sequences of the PVY(NA-N) and PVY(N:O) types were 99.0% and 99.5% identical to known sequences, respectively. There was no indication that recombinant strains PVY(NTN) or PVY(N:O) had different parental origins than recombinant strains previously sequenced. This is the first confirmation by whole-genome sequencing that "European"-type strain variants of PVY(N) and PVY(NTN) are present in North America, and the first reported full-length sequence of a tuber necrotic isolate of PVY(N:O).

Netrin-1 (Ntn-1) is a potent inducer of neuronal cell migration; however, its molecular mechanism that guides the migratory behavior of stem cells has not been characterized. In this study, we investigate the role of Ntn-1 in promoting the motility of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and its related signaling pathways. Ntn-1 (50 ng/mL) significantly increased motility of UCB-MSCs, which was inhibited by blocking antibodies for deleted in colorectal cancer (DCC) and integrin (IN) α6β4. Ntn-1 in DCC stimulated protein kinase Cα (PKCα) activation, but not PKCɛ, PKCθ, and PKCζ, while Ntn-1 in INα6β4 induced the phosphorylation of focal adhesion kinase (FAK) and Fyn. Notably, Ntn-1 induced phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and nuclear factor kappa-B (NF-κB), but they were concurrently downregulated by blocking the activities of PKCα, FAK, and Fyn. Ntn-1 uniquely increased the MMP-12 expression of all the matrix metalloproteinase (MMP) isoforms present in UCB-MSCs, though this was significantly blocked by an NF-κB inhibitor. Finally, Ntn-1 induced the MMP-12-dependent degradation of E-cadherin (E-cad), while Ntn-1 abrogated the interaction between E-cad and p120-catenin. In addition, Ntn-1 has the ability to stimulate cytoskeletal reorganization-related proteins, such as Cdc42, Rac1, Profilin-1, Cofilin-1, α-Actinin-4, and filamentous actin (F-actin) in UCB-MSCs. These results demonstrate that Ntn-1 induces MMP-12-dependent E-cad degradation via the distinct activation of PKCα and FAK/Fyn, which is necessary to govern the activation of ERK, JNK, and NF-κB in promoting motility of UCB-MSCs.

Inosine 5'-monophosphate (IMP) cyclohydrolase catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) to IMP in the final step of de novo purine biosynthesis. Two major types of this enzyme have been discovered to date: PurH in Bacteria and Eukarya and PurO in Archaea. The structure of the MTH1020 gene product from Methanothermobacter thermoautotrophicus was previously solved without functional annotation but shows high amino acid sequence similarity to other PurOs. We determined the crystal structure of the MTH1020 gene product in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) at 2.0 and 2.6 A resolution, respectively. On the basis of the sequence analysis, ligand-bound structures, and biochemical data, MTH1020 is confirmed as an archaeal IMP cyclohydrolase, thus designated as MthPurO. MthPurO has a four-layered alphabeta betaalpha core structure, showing an N-terminal nucleophile (NTN) hydrolase fold. The active site is located at the deep pocket between two central beta-sheets and contains residues strictly conserved within PurOs. Comparisons of the two types of IMP cyclohydrolase, PurO and PurH, revealed that there are no similarities in sequence, structure, or the active site architecture, suggesting that they are evolutionarily not related to each other. The MjR31K mutant of PurO from Methanocaldococcus jannaschii showed 76% decreased activity and the MjE102Q mutation completely abolished enzymatic activity, suggesting that these highly conserved residues play critical roles in catalysis. Interestingly, green fluorescent protein (GFP), which has no structural homology to either PurO or PurH but catalyzes a similar intramolecular cyclohydrolase reaction required for chromophore maturation, utilizes Arg96 and Glu222 in a mechanism analogous to that of PurO.

Spatial patterns of atmospheric deposition across the northeastern United States were evaluated and summarized in a simple model as a function of elevation and geographic position within the region. For wet deposition, 3-11 yr of annual concentration data for the major ions in precipitation were obtained from the National Atmospheric Deposition Program/National Trend Network (NADP/NTN) for 26 sites within the region. Concentration trends were evaluated by regression of annual mean concentrations against latitude and longitude. For nitrate, sulfate, and ammonium concentrations, a more than twofold linear decrease occurs from western New York and Pennsylvania to eastern Maine. These trends were combined with regional and elevational trends of precipitation amount, obtained from 30-yr records of annual precipitation at >300 weather stations, to provide long-term patterns of wet deposition. Regional trends of dry deposition of N and S compounds were determined using 2-3 yr of particle and gas concentration data collected by the National Dry Deposition Network (NDDN) and several other sources, in combination with estimates of deposition velocities. Contrary to wet deposition trends, the dominant air concentration trends were steep decreases from south to north, creating regional decreases in total deposition (wet + dry) from the southwest to the northeast. This contrast between wet and dry deposition trends suggests that within the northeast the two deposition forms are received in different proportions from different source areas, wet deposited materials primarily from areas to the west and dry deposited materials primarily from urban areas along the southern edge of the region. The equations generated describing spatial patterns of wet and dry deposition within the region were entered into a geographic information system (GIS) containing a digital elevation model (DEM) in order to develop spatially explicit predictions of atmospheric deposition for the region.

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.

There is increasing evidence that factors originally identified due to their neurotrophic activity also function within the immune system. This study focused on the related molecules glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) as well as their receptors. GDNF and NTN signaling is mediated by a two-component receptor: a signal-transducing component, RET, which is shared by both ligands, and a ligand-specific binding component, GFR alpha-1 (higher GDNF affinity) or GFR alpha-2 (higher NTN affinity). We report that human T cells, B cells, and monocytes produce NTN but not GDNF, as seen by RT-PCR and immunocytochemistry. RET was expressed by B cells, T cells, and monocytes. Exons 2-5 of RET encoding the cadherin-like domains 1-3 in the extracellular part and exons 16-19 encoding a section of the second tyrosine kinase domain were transcribed in CD4+ T cells, CD8+ T cells, B cells, and monocytes. Different splice variants encoding the C-terminal intracellular part (exons 19-21) of RET were detected. The ligand-binding receptors GFR alpha-1 and GFR alpha-2 were transcribed in all immune cell subsets. Quantitative PCR showed that GFR alpha-2 is by far the dominant ligand binding chain in T cells, B cells, and monocytes. Addition of GDNF or NTN to activated PBMCs reduced the amount of detectable TNF protein without altering its transcription. Together, this suggests that immune cells communicate with each other via NTN. Production of NTN by immune cells might also contribute to the neuroprotective immunity in the CNS observed in different model systems.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) mediate their actions through a unique multicomponent receptor system composed of Ret receptor tyrosine kinase and glycosyl-phosphatidylinositol-linked cell surface proteins (designated GFRalpha-1 and GFRalpha-2). In the present study, expression of these signalling components in the process of differentiation of haemopoietic cells was investigated. Ret was expressed at variable levels in normal and malignant cells of the myelomonocyte lineage. Immunohistochemical analysis of human and mouse tissues revealed that Ret expression was increased in intermediate mature myeloid cells such as promyelocytes and myelocytes and decreased in mature granulocytes and monocytes. Consistent with this observation, when THP-1 monocytic and HL-60 promyelocytic leukaemia cells expressing Ret were differentiated toward macrophages or granulocytes by treatment of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans retinoic acid (RA), Ret expression strikingly decreased during differentiation. Expression of GDNF, NTN, GFRalpha-1 and GFRalpha-2 was undetectable in THP-1 and HL-60 cells as well as in bone marrow haemopoietic cells. In contrast, bone marrow stromal cells appeared to express GDNF, GFRalpha-1 and GFRalpha-2 but not Ret. These findings suggested that the interaction between stromal cells and Ret-expressing haemopoietic cells in the bone marrow microenvironment may play a role in the differentiation of myelomonocyte-lineage cells through activation of the GDNF/Ret signalling pathway.

Intestinal ganglioneuromatosis (GN) is an uncommon disease of the enteric nervous system (ENS) and its pathogenesis remains unclear. Here we describe a unique case of diffuse GN of the intestinal wall associated with colon adenocarcinoma occurring in a 38-year-old female. Because it is well-known that glial cell line-derived neurotrophic factor (GDNF) and its receptor components, GDNF family receptor-alpha 1 (GFR-alpha 1) and RET receptor tyrosine kinase, play a crucial role in the development of ENS, their expression was analyzed by immunohistochemistry. Interestingly, GDNF as well as a related neurotrophic factor, neurturin (NTN), were expressed at high levels in adenocarcinoma cells whereas expression of GFR alpha 1 and RET was undetectable in them. In contrast, GFR-alpha 1 showed positive staining in both proliferating ganglion cells and glial cells, and RET immunoreactivity was found mainly in ganglion cell bodies. These findings suggested that GDNF and NTN expression in adenocarcinoma cells may play an important role in the pathogenesis of GN.

Breast cancer is a disease affecting an increasing number of women worldwide. Several efforts have been made in the last years to identify imaging biomarker and to develop noninvasive diagnostic tools for breast tumor characterization and monitoring, which could help in patients' stratification, outcome prediction, and treatment personalization. In particular, radiomic approaches have paved the way to the study of the cancer imaging phenotypes. In this work, a group of 49 patients with diagnosis of invasive ductal carcinoma was studied. The purpose of this study was to select radiomic features extracted from a DCE-MRI pharmacokinetic protocol, including quantitative maps of ktrans, kep, ve, iAUC, and R1 and to construct predictive models for the discrimination of molecular receptor status (ER+/ER-, PR+/PR-, and HER2+/HER2-), triple negative (TN)/non-triple negative (NTN), ki67 levels, and tumor grade. A total of 163 features were obtained and, after feature set reduction step, followed by feature selection and prediction performance estimations, the predictive model coefficients were computed for each classification task. The AUC values obtained were 0.826 ± 0.006 for ER+/ER-, 0.875 ± 0.009 for PR+/PR-, 0.838 ± 0.006 for HER2+/HER2-, 0.876 ± 0.007 for TN/NTN, 0.811 ± 0.005 for ki67+/ki67-, and 0.895 ± 0.006 for lowGrade/highGrade. In conclusion, DCE-MRI pharmacokinetic-based phenotyping shows promising for discrimination of the histological outcomes.

Many bacteria, such as Proteobacteria, Cyanobacteria and Bacteroidetes, use N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules for communication. Enzymatic degradation of AHLs, such as AHL acylase and AHL lactonase, can degrade AHLs (quorum quenching, QQ) to attenuate or disarm the virulence of pathogens. QQ is confirmed to be common in marine bacterial communities. Many genes encoding AHL acylases are found in marine bacteria and metagenomic collections, but only a few of these have been characterized in detail. We have reported that the marine bacterium Pseudoalteromonas flavipulchra JG1 can degrade AHLs. In the present study, a novel AHL acylase PfmA, which can degrade AHLs with acyl chains longer than 10 carbons, was identified from strain JG1. Ultra-performance liquid chromatography (UPLC) and electrospray ionization mass spectrometry (ESI-MS) analysis demonstrated that PfmA functions as an AHL acylase, which hydrolysed the amide bond of AHL. The purified PfmA of P. flavipulchra JG1 showed optimum activity at 30 °C and pH 7.0. PfmA belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily and showed homology to a member of penicillin amidases, but PfmA can degrade ampicillin but not penicillin G. The residue Ser256 in PfmA is the active site according to site-directed mutagenesis. Furthermore, PfmA reduced AHL accumulation and the production of virulence factors in Vibrio anguillarum VIB72 and Pseudomonas aeruginosa PAO1, and attenuated the virulence of P. aeruginosa to increase Artemia survival, which suggested that PfmA can be considered as a therapeutic agent to control AHL-mediated pathogenicity.

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY(O)). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY(O) whilst failing to detect three isolates of PVY(N) and 12 isolates of PVY(NTN). Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vlambda1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.

This report describes the characterization by whole-genome sequencing of four PVY isolates with unique combinations of molecular and symptomatic characteristics. Three of these four isolates were of type PVY(N:O) (ID-1, OR-1, PN10A), including one of "type B", which contains an extra recombination event in the 5'UTR/P1 cistron; the other (NE-11) represents a novel PVY molecular genotype, previously misclassified as a PVY(NA-NTN) isolate. The full genome sequence of this latter isolate is unique inasmuch as it is nearly identical to that of PVY(N) isolates for the first 2,000 nucleotides (nts), after which it very strongly resembles PVY(NA-NTN) isolates for the next 600 nts. For the final 7,000 nts of its genome, NE-11 shares intermediate identity with these other two previously reported classes of PVY(N) genomes, except for a portion of the capsid protein region in which it resembles neither. Recombination in each of the four isolates was verified by a suite of recombination detection programs. PN10A represents the first complete sequence of a PVY strain variant of the class reported as PVY(N)-W (or PVY(N:O)) type B. Specific PCR assays for two unique regions of NE-11 are presented that will allow the identification of this strain variant by other researchers.