La concentration plasmatique de PTH permet-elle de sélectionner les patients atteints d’hyperparathyroïdie secondaire pour bénéficier de la scintigraphie double isotope MIBI (99mTc)/123I préopératoire ?

The utility of preoperative scintigraphy in case of secondary hyperparathyroidism is questioned by some authors. Obviously, an imaging modality that will detect all hyperplastic glands, including the ectopic ones, would be of interest in those patients at high risk for surgery. However, scintigraphy has a limited detection rate in some patients. We investigated whether one of the following parameters would identify a subgroup of patients in whom the detection rate would be optimal: age, gender, hemodialysis and duration since its onset, and plasma levels of parathyrin (PTH).MethodsRetrospective series of 38 patients referred for preoperative parathyroid scintigraphy due to secondary hyperparathyroidism who then underwent parathyroidectomy. Scintigraphy was performed 20 min and then 3 h after injection of 8 MBq/kg of sestamibi (^99mTc) with a previous ingestion of 0.1 MBq/kg iodine-123, 3 h before.ResultNo significant correlation was observed between the number of glands detected on scintigraphy (and confirmed by postoperative histology) and plasma PTH levels (r = ?0.17). A weak positive correlation (r = +0.34) was noted in the group of six non-hemodialysed patients. No significant relationship between this number of detected glands and a clinical parameter was observed.ConclusionIn our experience, these parameters do not permit to select, among patients with secondary hyperparathyroidism and scheduled for parathyroidectomy, those who will better benefit from parathyroid scintigraphy.     

Survey of medical training in cytopathology carried out by the journal Cytopathology

Anshu, A. Herbert, B. Cochand‐Priollet, P. Cross, M. Desai, R. Dina, J. Duskova, A. Evered, A. Farnsworth, W. Gray, S. S. Gupta, K. Kapila, I. Kardum‐Skelin, V. Kloboves‐Prevodnik, T. K. Kobayashi, H. Koutselini, W. Olszewski, B. Onal, M. B. Pitman, ?. Marin?ek, T. Sauer, U. Schenck, F. Schmitt, I. Shabalova, J. H. F. Smith, E. Tani, L. Vass, P. Vielh and H. Wiener
Survey of medical training in cytopathology carried out by the journal Cytopathology This report of the Editorial Advisory Board of Cytopathology gives the results of a survey of medical practitioners in cytopathology, which aimed to find out their views on the current situation in undergraduate and postgraduate training in their institutions and countries. The results show that training in cytopathology and histopathology are largely carried out at postgraduate level and tend to be organized nationally rather than locally. Histopathology was regarded as essential for training in cytopathology by 89.5% of respondents and was mandatory according to 83.1%. Mandatory cytopathology sections of histopathology were reported by 67.3% and specific examinations in cytopathology by 55.4%. The main deficiencies in training were due to its variability; there were insufficient numbers of pathologists interested in cytology and a consequent lack of training to a high level of competence. Pathologists without specific training in cytopathology signed out cytology reports according to 54.7% of responses, more often in centres where training was 3–6 months or less duration. Although 92.2% of respondents thought that specialist cytology should not be reported by pathologists without experience in general cytopathology, that practice was reported by 30.9%, more often in centres with small workloads. The survey report recommends that 6–12 months should be dedicated to cytopathology during histopathology training, with optional additional training for those wanting to carry out independent practice in cytopathology. Formal accreditation should be mandatory for independent practice in cytopathology. When necessary, temporary placements to centres of good practice should be available for trainees intending to practise independently in cytopathology. There should be adequate numbers of pathologists trained in cytopathology to a high level of competence; some of their time could be released by training cytotechnologists and trainee pathologists to prescreen cytology slides and assess adequacy of fine‐needle aspiration samples when immediate diagnosis was not required. The survey demonstrated a clear need for European and international guidelines for training in cytopathology.     

Mycorrhizal association and symbiotic germination of the terrestrial orchid Bipinnula fimbriata (Poepp.) Johnst (Orchidaceae)

In this study mycorrhizal fungi were isolated from the roots of the endemic terrestrial orchid Bipinnula fimbriata. Seven isolates were previously identified as the form-genus Rhizoctonia, a polyphyletic group known to form mycorrhizal associations with Orchidaceae. Two other isolates were included in the study: #793 isolated from Chloraea crispa, and #1325 Rhizoctonia solani, isolated from potato. After morphological and molecular characterization of the nine isolates, they were divided into three groups, Ceratobasidium sp., Tulasnella calospora and Thanatephorus cucumeris, to determine the diversity between isolates. Consensus ITS sequences were used for a blast search on the GenBank database, which confirmed the results of the morphological observations. Once the isolates were identified, an in vitro germination test was done with four plates of oatmeal agar inoculated with each fungus, plus an asymbiotic control. The germination stages of the seeds were recorded 30 days after sowing. All isolates obtained from B. fimbriata, and the isolate #793 from Chloraea crispa, promoted seed germination. However, the isolate #1325 Rhizoctonia solani, which is known as both a pathogen and an orchid symbiont, did not promote germination. This shows that B. fimbriata is associated with more than one mycorrhizal fungus in its habitat and has a broader potential specificity in vitro. The results support the hypothesis that at least one fungal isolate promotes the germination of B. fimbriata, permitting the conservation of this species in ex situ conditions.     

Neuropoietin activates STAT3 independent of LIFR activation in adipocytes

Neuropoietin (NP) is a member of the gp130 cytokine family that is closely related to cardiotrophin-1(CT-1) and shares functional and structural features with other family members, including ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC). Studies have shown that NP can play a role in the development of the nervous system, as well as affect adipogenesis and fat cell function. However, the signaling mechanisms utilized by NP in adipocytes have not been examined. In our present studies, we demonstrate that NP-induced activation of STAT3 tyrosine phosphorylation is independent of leukemia inhibitory factor receptor (LIFR) phosphorylation and degradation. Although it is widely accepted that NP signals via the LIFR, our studies reveal that NP results in phosphorylation of gp130, but not LIFR. These observations suggest that the profound effects that NP has on adipocytes are not mediated via LIFR signaling.     

Genome‐scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production in Pichia pastoris

A method combining fluorescence activated cell sorting (FACS) and DNA microarray assisted clone identification was developed and termed Genome‐Scale Analysis of Library Sorting (GALibSo). Genes enhancing the production of secreted heterologous proteins in Pichia pastoris were identified out of a cDNA library by cell surface display and FACS. The trends of gene enrichment during consecutive FACS rounds were monitored by DNA microarrays. In a case study a P. pastoris cDNA library was co‐expressed in a strain secreting the Fab fragment of a monoclonal antibody against human immunodeficiency virus type 1 as a model protein. Three genes were identified, increasing the relative expression level of the surface‐displayed model protein up to 45%. While one of these genes had a positive effect on three out of four tested proteins, the product specific effect of the other two suggested that the effects of the co‐expressed secretion enhancing factors are partly dependent on the protein to be produced. The microarray based monitoring of the enrichment of genes causing enhanced protein secretory capacity led to novel insights into the limitation of protein secretion. Biotechnol. Bioeng. 2010; 105: 543–555. © 2009 Wiley Periodicals, Inc.     

Role of Annexin A2 in the Production of Infectious Hepatitis C Virus Particles

Hepatitis C virus (HCV) is an important human pathogen affecting 170 million chronically infected individuals. In search for cellular proteins involved in HCV replication, we have developed a purification strategy for viral replication complexes and identified annexin A2 (ANXA2) as an associated host factor. ANXA2 colocalized with viral nonstructural proteins in cells harboring genotype 1 or 2 replicons as well as in infected cells. In contrast, we found no obvious colocalization of ANXA2 with replication sites of other positive-strand RNA viruses. The silencing of ANXA2 expression showed no effect on viral RNA replication but resulted in a significant reduction of extra- and intracellular virus titers. Therefore, it seems likely that ANXA2 plays a role in HCV assembly rather than in genome replication or virion release. Colocalization studies with individually expressed HCV nonstructural proteins indicated that NS5A specifically recruits ANXA2, probably by an indirect mechanism. By the deletion of individual NS5A subdomains, we identified domain III (DIII) as being responsible for ANXA2 recruitment. These data identify ANXA2 as a novel host factor contributing, with NS5A, to the formation of infectious HCV particles.Hepatitis C virus (HCV) infections are characterized by a mostly unapparent acute phase leading to persistence in ca. 70% of all infected individuals. Currently, 170 million people suffer from chronic hepatitis C, and they have a high risk to develop severe liver disease. It has been estimated that HCV accounts for 27% of cirrhosis and 25% of hepatocellular carcinoma cases worldwide (2).HCV is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae. The genome of HCV encompasses a single ∼9,600-nucleotide (nt)-long RNA molecule containing one large open reading frame (ORF) that is flanked by nontranslated regions (NTRs), which are important for viral translation and replication. HCV proteins generated from the polyprotein precursor are cleaved by cellular and viral proteases into at least 10 different products (for a review of polyprotein cleavage and the function of the individual proteins, see reference 4). The structural proteins Core, E1, and E2 are located in the amino-terminal portion of the polyprotein, followed by p7, a hydrophobic peptide that is supposed to be a viroporin, and the nonstructural proteins (NS) NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Only the nonstructural proteins NS3 to NS5B are involved in viral RNA replication. NS3 is a multifunctional protein, consisting of an amino-terminal protease domain required for the processing of the NS3 to NS5B region and a carboxyterminal helicase/nucleoside triphosphatase domain. NS4A is a cofactor that activates the NS3 protease function by forming a heterodimer. The hydrophobic protein NS4B induces vesicular membrane alterations involved in RNA replication. NS5A is a phosphoprotein that seems to play an important role in viral replication and assembly (3, 35, 58). NS5B is the RNA-dependent RNA polymerase of HCV.Positive-strand RNA viruses replicate their RNA in vesicular structures originating from different cellular organelles (36). In the case of HCV, particular membrane alterations have been identified by electron microscopy, designated the membranous web, consisting of accumulations of vesicles primarily derived from the endoplasmic reticulum (17). Important insights into the organization of HCV replication complexes were obtained by the in vitro analysis of viral RNA synthesis in membrane preparations of cells harboring subgenomic HCV replicons, so-called crude replication complexes (CRCs) (1, 20). A current model based on a stoichiometric analysis of CRCs suggests that each vesicular structure contains multiple copies of viral nonstructural proteins and has a connection to the cytoplasm, allowing the constant supply of nucleotides for RNA synthesis (45), presumably analogously to the replication complex of the closely related dengue virus (DV) (64). Viral RNA synthesis in CRCs is highly resistant to proteinases and nucleases (39), and the membranes are detergent resistant at 4°C, resembling features of lipid rafts (54).Several purification techniques have been established to identify relevant HCV host factors by proteomics, based on either the extraction of detergent-resistant membranes (19, 34) or the immunoprecipitation of vesicles (24), revealing different sets of cellular proteins potentially involved in viral replication. In most of these studies, cell lines harboring persistent subgenomic replicons were utilized (33); however, with the availability of a fully permissive cell culture system supporting the complete HCV replication cycle (31, 63, 66), it became evident that viral RNA replication and assembly are closely linked. Recent work revealed an intimate connection of viral replication complexes and assembly sites in close proximity to cytoplasmic lipid droplets (38), with Core and especially NS5A functioning as central regulators by a poorly defined mechanism. NS5A is phosphorylated at multiple serine and threonine residues, binds RNA, and is composed of three domains, which are separated by trypsin-sensitive low-complexity regions (LCS I and II) (59). An N-terminal amphipathic alpha helix tightly associates NS5A with intracellular membranes. Domain I and LCS1 most likely are involved in viral RNA replication, since replication-enhancing mutations primarily mapped to this region (8, 32). The role of domain II is unknown, while domain III recently has been shown to be dispensable for RNA replication but essential for viral particle assembly (3, 35, 58). One of the proposed mechanisms points to a critical interaction with the Core protein, for which phosphorylation in the C-terminal part of domain III of NS5A appears to be required (35). The interaction of Core and NS5A has been proposed to be important for the recruitment of the replication complexes to lipid droplets (3), thereby allowing a coordinated packaging of the newly synthesized RNA.In this study, we identified annexin A2 (also called annexin II, calpactin 1, and ANXA2) as an HCV host factor by a proteomic analysis. ANXA2 belongs to a family of proteins characterized by their Ca²⁺-dependent binding to negatively charged phospholipids. The annexin proteins consist of two principle domains, a variable N-terminal and a conserved C-terminal domain, which harbors the Ca²⁺ and membrane binding sites (for a review, see references 14 and 15). All annexins show cytosolic and membrane localizations. Membrane recruitment probably is regulated by intracellular Ca²⁺ fluctuations, and target membrane selection differs for different annexins.In addition to showing a cytosolic distribution, ANXA2 can associate with the plasma membrane and the membrane of early endosomes. Plasma membrane-associated ANXA2 typically is found in a tight heterotetrameric complex with the S100 protein S100A10 (p11). ANXA2 specifically interacts with phosphatidylinositol(4,5)bisphosphate (PIP2) (22, 48) and binds to membranes enriched in cholesterol, supporting a role in the organization of lipid raft-like membrane microdomains. Due to the direct binding of ANXA2 to F-actin, the protein has been proposed to provide a direct link between cytoskeletal elements and PIP2/cholesterol-rich membrane domains (47).ANXA2 has been implicated in several cellular transport processes, including the internalization and transport of cholesteryl esters, the biogenesis of multivesicular bodies, the recycling of plasma membrane receptors, and the Ca²⁺-induced exocytosis of certain secretory granules (14). Here, we show that ANXA2 is present at HCV replication sites within the membranous web. The recruitment of ANXA2 is mediated by domain III of NS5A and probably is required for efficient virus assembly.     

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome encodes several smaller open reading frames (ORFs) located in the 3′ region of the genome that are predicted to express eight novel proteins termed accessory proteins. The accessory proteins are designated ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b and range in size from 39 to 274 amino acids (35, 50). These SARS-CoV-specific ORFs are not present in other coronaviruses and do not display significant homology with any known proteins in the NCBI database. Five of these are predicted to code for polypeptides of greater than 50 amino acids (35, 50). Antibodies reactive against all of the SARS-CoV proteins have been detected in sera isolated from SARS patients, indicating that these proteins are expressed by the virus in vivo (7, 9, 17-19, 45, 59). Expression of three of the ORF proteins has been demonstrated during infection using protein-specific antibodies and include the ORFs 3a, 6, and 7a (12, 37, 41, 60). Six of the eight group-specific ORFs, including ORFs 3a, 3b, 6, 7a, 7b, and 9b, were deleted from recombinant SARS-CoV and shown to be dispensable for in vitro and in vivo replication (66).Related coronaviruses also encode unique accessory proteins in the 3′ region of the genome, often referred to as group-specific ORFs. Similar to SARS-CoV, several of these proteins are dispensable for viral replication. Murine hepatitis virus (MHV) expresses accessory proteins ORFs 2a, 4, and 5a. A recombinant virus in which ORF 2a was deleted replicated normally in vitro but caused attenuated disease in vivo (55). Deletion of the group-specific ORF 7 in porcine coronavirus TGEV also results in reduced replication and virulence in vivo despite normal replication in vitro (38). Similarly, in feline infectious peritonitis virus (FIPV), group-specific proteins are dispensable for replication in cell culture but contribute to pathogenesis in vivo (20). Thus, while the SARS-CoV group specific proteins are unnecessary for in vitro and in vivo replication, their expression may underlie the devastating pathology associated with SARS disease. Detailed characterization of these novel proteins may contribute to a better understanding of SARS pathogenesis and host-virus interactions.The ORF 3a protein is expressed from subgenomic RNA3, which contains the 3a and 3b ORFs (35, 50). The 3a protein, which is the largest group-specific SARS-CoV accessory protein at 274 amino acids, has been reported to localize to the Golgi apparatus, the plasma membrane, and intracellular vesicles of unknown origin (67, 68). The protein is efficiently transported to the cell surface and is also internalized during the process of endocytosis (60).The mechanism of SARS-CoV-induced cell death has been investigated by several groups. Studies to date have used overexpression of individual SARS-CoV ORFs to evaluate their intrinsic cytotoxicity. Using this approach, the following proteins have been reported to cause apoptosis: the 3CL-like protease; spike; ORFs 3a, 3b, and 7a; and the envelope (E), membrane (M), and nucleocapsid (N) proteins (23, 31, 32, 36, 46, 58, 61, 65, 69). However, since all of these reports utilize overexpression of individual proteins, it is unclear whether these effects may be attributable to high, nonphysiological levels of protein and whether they occur during infection. Analysis of recombinant viruses with specific mutations or deletions is necessary to determine the relative contribution of these proteins to the cytotoxicity of SARS-CoV during infection (63). Therefore, the cytotoxic component(s) of SARS-CoV have not been fully defined.Here, we have investigated the function of the ORF 3a protein in the context of SARS-CoV infection and by overexpression. We confirm that ORF 3a contributes to SARS-CoV cytotoxicity using a recombinant strain deficient for expression of ORF 3a. While characterizing this deficient strain, we observed that SARS-CoV-induced vesicle formation, a feature that has been documented in cells from infected SARS patients, is dependent on ORF 3a. Furthermore, we observed that SARS-CoV infection causes Golgi fragmentation by ORF 3a. Additional characterization of 3a in transfected cells revealed that the protein colocalizes with markers of the trans-Golgi network (TGN) and late endosomal pathways and causes an accumulation of these vesicles. Finally, we report that Arf1 overexpression rescued SARS-CoV or 3a-induced Golgi fragmentation, suggesting that the ORF 3a protein may perturb Arf1-mediated vesicle trafficking.     

Multiple Sequence Signals Direct Recognition and Degradation of Protein Substrates by the AAA+ Protease HslUV

Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V_max. By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease.