High performance of a novel antigen detection test on nasopharyngeal specimens for diagnosing SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has become a major public health issue worldwide. Developing and evaluating rapid and easy-to-perform diagnostic tests is a high priority. The current study was designed to assess the diagnostic performance of an antigen-based rapid detection test (COVID-VIRO®) in a real-life setting. Two nasopharyngeal specimens of symptomatic or asymptomatic adult patients hospitalized in the Infectious Diseases Department or voluntarily accessing the COVID-19 Screening Department of the Regional Hospital of Orléans, France, were concurrently collected. The diagnostic specificity and sensitivity of COVID VIRO® results were compared to those of real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) results. A subset of patients underwent an additional oropharyngeal and/or saliva swab for rapid testing. A total of 121 patients confirmed to be infected and 127 patients having no evidence of recent or ongoing infection were enrolled for a total of 248 nasopharyngeal swab specimens. Overall, the COVID-VIRO® sensitivity was 96.7% (CI, 93.5%-99.9%). In asymptomatic patients, symptomatic patients having symptoms for more than 4 days and those with an RT-qPCR cycle threshold value ≥ 32, the sensitivities were 100%, 95.8%, and 91.9%, respectively. The concordance between RT-qPCR and COVID VIRO® rapid test results was 100% for the 127 patients with no SARS-CoV-2 infection. The COVID-VIRO® test had 100% specificity and sensitivity greater than 95%, which are better than the recommendations set forth by the WHO (specificity ≥ 97%-100%, sensitivity ≥ 80%). These rapid tests may be particularly useful for large-scale screening in emergency departments, low-resource settings, and airports.

Optical properties of functionalized nanodiamonds.

Detonation nanodiamonds exhibit strong nonlinear optical properties depending on their electronic properties. In the present paper, the nanodiamond functional groups are chemically modified to obtain nanodiamonds with primary amines on their surface. The optical properties of such nanodiamonds placed in water suspensions are studied and compared with the one of classical detonation nanodiamonds. Transmission, scattering and Z-scan experiments are performed for two different wavelengths (532 nm and 1064 nm). A lower threshold for optical limiting associated to more pronounce non-linear optical effects is detected at the wavelength of 1064 nm compared to the one at 532 nm. This effect may be due to a stronger nonlinear backscattering behavior at 1064 nm. Moreover, a striking result obtained from the Z-scan experiments reveals a completely different behavior of the functionalized nanodiamonds for both wavelengths. This result is discussed in regard to the electronic properties of the material and possible charge transfer.

Non polymeric nanoparticles for photodynamic therapy applications: recent developments.

Photodynamic therapy has emerged as an alternative to chemotherapy and radiotherapy for cancer treatment. Nanoparticles have recently been proposed as effective carriers for photosensitizers. Depending on their chemical composition, these can be used for diagnosis and therapy due to the selective accumulation of the photosensitizer in cancer cells in vitro or in tumors in vivo. Multifunctional nanoplatforms combining several applications within the same nano-object emerge as potential important theranostic tools. This review, based on the chemical nature of the nanoparticles will discuss recent advances in the area of non polymeric nanoparticles for photodynamic therapy applications.

The cellular prion protein: a new partner of the lectin CBP70 in the nucleus of NB4 human promyelocytic leukemia cells.

Prion diseases are characterized by the presence of an abnormal isoform of the cellular prion protein (PrPc) whose physiological role still remains elusive. To better understand the function of PrPc, it is important to identify the different subcellular localization(s) of the protein and the different partners with which it might be associated. In this context, the PrPc-lectins interactions are investigated because PrPc is a sialoglycoprotein which can react with lectins which are carbohydrate-binding proteins. We have previously characterized a nuclear lectin CBP70 able to recognize N-acetyl-beta-D-glucosamine residues in HL60 cells. Using confocal immunofluorescence, flow-cytofluorometry, and Western-blotting, we have found that PrPc is expressed in the nucleus of the NB4 human promyelocytic leukemia cell line. It was also found that the lectin CBP70 is localized in NB4 cell nuclei. Moreover, several approaches revealed that PrPc and CBP70 are colocalized in the nucleus. Immunoprecipitation experiments showed that these proteins are coprecipitated and interact via a sugar-dependent binding moiety. In conclusion, PrPc and CBP70 are colocalized in the nuclear compartment of NB4 cells and this interaction may be important to better understand the biological function and possibly the conversion process of PrPc into its pathological form (PrPsc).

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?

Stable expression of functional CBP70 lectin during heat shock.

CBP70 is a glycoslylated lectin that interacts through either glycan-lectin or protein-protein interactions. In addition, depending on its cellular localization, this lectin has different partners, for example, galectin-3, an 82-kDa ligand in the nucleus, or Bcl-2 in the cytoplasm. In this study, we observed the persistence of plurilocalized lectin CBP70 after two heat-shock treatments conducted either under mild conditions, i.e., incubating the cells for 1 h at 42 degrees C then for 1, 3, 5, 7, or 9 h at 37 degrees C, or harsh conditions, i.e., incubation at 42 degrees C for 1, 2, 4, 6, 8, or 10 h. By combining the information collected from biochemical, fluorocytometric, confocal, and affinity-chromatography analyses, we concluded that CBP70 persisted in HL60 cells and its N-acetylglucosamine-binding sites remained active after all the heat-shock treatments tested. These data and the previously published findings reviewed in this report concur in supporting the hypothesis that CBP70 could function as an organizer of multimeric assembly, leading to the formation of various complexes in different cellular compartments, according to the needs of the cell.

Glycoaffinity chromatography and biological recognition.

The potential of bioaffinity chromatography as a tool for study of biological recognition mechanisms is gaining increasing recognition. Biochromatographic methods allow the separation of proteins according to both the structure of their polypeptidic chain and their post-translational modifications. Among the various post-translational modifications which proteins undergo, glycosylation has conducted to the development of original methods (glycotechnologies). This review discusses the applications of glycotechnologies in bioaffinity chromatography, and particularly the use of biochromatography to elucidate mechanisms involved in glycobiology.

CBP70, a glycosylated nuclear lectin.

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.

Characterization of a putative 82 kDa nuclear ligand for the N-acetylglucosamine-binding protein CBP70.

Since nuclear lectins were first characterized several years ago, six lectins have been isolated. Furthermore, the existence of nuclear glycoproteins containing N-linked complex-oligosaccharide chains or O-linked GlcNAc residues was evidenced. These latter are abundant in the nucleus and are well-studied so far. The presence of both glycoprotein and lectin in the cell nucleus led us to postulate that these two proteins could interact and play a role in some nuclear activities such as the modulation of transcription and/or nuclear cytoplasmic exchanges or by the disruption of protein-protein interactions. In such context, the recent data concerning the GlcNAc-binding activity of CBP70 argued this postulate. However, to study the possible role of a glycoprotein-lectin complex, it was critical to isolate the two partners. Because CBP70 was also a cytoplasmic protein, the lectin was isolated in both cytoplasmic and nuclear compartments in order to investigate the putative ligand in the two cellular compartments. The results obtained with cross-linking experiments on isolated and membranedepleted nuclei incubated with the CBP70 bearing an iodinatable, cleavable, photoreactive cross-linking agent (sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate) and immunoprecipitation experiments with polyclonal antibodies raised against CBP70, revealed that both nuclear and cytoplasmic CBP70 have the same 82 kDa nuclear ligand which is absent in the cytoplasmic fraction. In addition, this ligand is glycosylated, containing GlcNAc residues, and, therefore, the complex between CBP70 and the 82 kDa polypeptide could be due to a glycoprotein-lectin interaction. These results raised the possibility that nuclear glycoprotein-lectin interaction could be involved in nuclear activities.

Nuclear and cytoplasmic expressions of the carbohydrate-binding protein CBP70 in tumoral or healthy cells of the macrophagic lineage.

The expression of the carbohydrate-binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte-derived macrophages (MDM) using an anti-CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N-acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60-derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti-CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 and p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin-3.

Intranuclear distribution of galectin-3 in mouse 3T3 fibroblasts: comparative analyses by immunofluorescence and immunoelectron microscopy.

The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli.

Expression of nuclear lectin carbohydrate-binding protein 35 in human immunodeficiency virus type 1-infected Molt-3 cells.

The nuclear carbohydrate-binding protein 35 (CBP35), a beta-galactoside-specific lectin with an M(r) of 35,000, has been identified in nuclear ribonucleoprotein complexes (RNPs) from a variety of mammalian tissues and cells. Here we determined that the expression of CBP35 mRNA greatly increases after infection of Molt-3 cells with human immunodeficiency virus type 1 (HIV-1), concomitantly with the onset of expression of the viral regulatory gene tat, and then declines. The increase in CBP35 mRNA level results in an enhanced synthesis of CBP35, as evidenced in nitrocellulose filter binding assay using radiolabeled, sugar-specific neoglycoprotein. Immunoblotting experiments showed that CBP35 is present in the 40S heterogeneous nuclear RNP complex from HIV-1-infected Molt-3 cells. CBP35 could also be detected using a novel photoreactive alpha-D-galactose probe designed for the specific detection of CBP.

Identification of two nuclear N-acetylglucosamine-binding proteins.

Using neoglycoproteins, lectins that recognize different sugars, including N-acetylglucosamine residues, were previously detected in animal cell nuclei. We report herein the isolation of two N-acetylglucosamine-binding proteins from HL60 cell nuclei: i) a 22 kDa polypeptide (CBP22) with an isoelectric point of 4.5 was isolated for the first time and ii) a 70 kDa polypeptide with an isoelectric point of 7.8. This latter protein corresponds to the glucose-binding protein (CBP70) previously isolated, based on the following similarities: i) they have the same molecular mass, ii) they have the same isoelectric point, iii) they are recognized by antibodies raised against CBP70, and iv) both are lectins from the C group of Drickamer's classification. CBP70 appeared to recognize glucose and N-acetylglucosamine; however, its affinity for N-acetylglucosamine was found to be twice that for glucose. The presence in the nucleus of two nuclear N-acetylglucosamine-binding proteins and their potential ligands, such as O-N-acetylglucosamine glycoproteins, strongly argues for possible intranuclear glycoprotein-lectin interactions.

Evidence that lactose binding to CBP35 disrupts its interaction with CBP70 in isolated HL60 cell nuclei.

We have previously reported that two carbohydrate-binding proteins (CBP35 and CBP70) can, under appropriate conditions of affinity chromatography, be isolated from HL60 cell nuclear extracts as a complex. Moreover, we have demonstrated that, during affinity chromatography, the CBP70-CBP35 association can be modified by the binding of lactose to CBP35. To determine whether the CBP70-CBP35 association could be disrupted in the nucleus upon lactose binding to CBP35, the behaviors of CBP70 and CBP35 were analyzed in membrane-depleted nuclei of HL60 cells, incubated with or without lactose. This study was performed by using an antiserum that cross-reacts with CBP35 and CBP70, an antiserum that was specifically raised against CBP70, and a monoclonal antibody (Mac 2) reactive against CBP35. Taken together, the results of indirect immunofluorescent staining, immunoblotting experiments, and quantitative flow-cytofluorometric analysis show that (i) CBP70 and CBP35 are present and colocalized in the nuclei incubated without lactose, (ii) all the CBP70 molecules left the nuclei incubated in the presence of lactose, while CBP35 molecules, probably bound to RNA, remained inside the nuclei, and (iii) glucose failed to have the same effect as lactose. These results strongly suggest that, in membrane-depleted nuclei, CBP35 and CBP70 interactions can be altered by a conformational change of CBP35 induced by the binding of lactose to its carbohydrate-recognition domain.

[CBP35-CBP67 interaction in stress response and aging]. / CBP35-CBP67-Interaktion im Verlauf der Stressantwort und des Alterns.

Three carbohydrate-binding proteins with relative molecular masses of 35, 67, and 70 kDa (CBP35, CBP67, and CBP70) have been described to be present in nuclei of mammalian cells, where they are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an N-terminal domain that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a C-terminal domain that is homologous to beta-galactoside-specific lectins. CBP35 has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here, we show that exposition of mature rats (6-8 months old) to stress results in binding of nuclear CBP35 to CBP67 which is retained on a column containing immobilized glucose. In contrast to mature animals, nuclear extracts from the livers of old rats (22-24 months old) displayed no detectable stress response.

The human HIP gene, overexpressed in primary liver cancer encodes for a C-type carbohydrate binding protein with lactose binding activity.

HIP was originally identified as a gene expression in primary liver cancers, and in normal tissues such as pancreas and small intestine. Based on gene data base homologies, the HIP protein should consist of a signal peptide linked to a single carbohydrate recognition domain. To test this hypothesis HIP and the putative carbohydrate recognition domain encoded by the last 138 C-terminal amino acids, were expressed as glutathione-S-transferase proteins (GST-HIP and GST-HIP-142, respectively). Both recombinant proteins were purified by a single affinity purification step from bacterial lysates and their ability to bind saccharides coupled to trisacryl GF 2000M were tested. Our results show that HIP and HIP-142 proteins bind to lactose, moreover the binding requires divalent cations. Thus the HIP protein is a lactose-binding lectin with the characteristics of a C-type carbohydrate recognition domain of 138 amino acids in the C-terminal region.

HnRNP CBP35.CBP67 interaction during stress response and ageing.

Previous studies have demonstrated the existence of nuclear carbohydrate binding proteins in a variety of mammalian cells with molecular masses of 35,000, 67,000, and 70,000 (CBP35, CBP67, and CBP70), which are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an amino-terminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a carboxyl-terminal portion homologous to beta-galactoside-specific lectins. CBP35 it has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here we show that the exposure of mature rats to stress induces an increase in nuclear CBP35 bound to CBP67 and retained on immobilized glucose. Nuclear extracts from the livers of old rats displayed no detectable stress response. This CBP35.CBP67 association detected in rat liver is considered with respect to the CBP35.CBP70 association recently observed in HL60 cell nuclear extracts.

[Age-dependent changes in mRNA transport (nucleus-cytoplasm)]. / Alternsabhängige Veränderungen des mRNA-Transports (Kern-Zytoplasma).

Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase. In addition, we found that an RNA helicase activity is present in NE, which also may be involved in RNA transport. Our results show that, besides poly(A), also double-stranded RNA structures may modulate RNA export. The amount of mRNA released from nuclei markedly decreases with age. Evidence is presented that this age-dependent change is caused by an impairment of polyadenylation of mRNA, hnRNA processing, release of mRNA from nuclear matrix, and translocations of mRNA from nuclear to cytoplasmic compartment (decrease in activities of NE NTPase, protein kinase, and phosphatase; decrease in poly(A)-binding affinity of mRNA carrier).

Evidence for a lactose-mediated association between two nuclear carbohydrate-binding proteins.

Nuclear proteins were extracted in 2 M NaCl from membrane-depleted nuclei isolated from HL60 cells. Extracted proteins were submitted to affinity chromatography columns containing immobilized glucose, galactose or lactose. The polypeptides present in the different eluted fractions were resolved by SDS-PAGE and were either silver stained or analysed by immunoblotting with monoclonal or polyclonal antibodies, respectively, raised against the glucose-binding protein CBP67 and the galactose-binding proteins CBP35 and L14. The results presented here show that HL60 cell nuclei contain CBP35 and a glucose-binding lectin of 70 kDa (CBP70). These data account for the previously reported binding of neoglycoproteins containing glucosyl and galactosyl residues to HL60 cell nuclei. Furthermore, the present study provides the new information that CBP35 can associate with CBP70 by interactions dependent on the binding of CBP35 to lactose, and the results of some affinity chromatography experiments strongly suggest that CBP35 and CBP70 associate by protein-protein interactions. The potential function of this lactose-mediated interaction is discussed with respect to data recently reported by others showing that CBP35 is involved in in vitro mRNA splicing and that lactose inhibits the processing of the pre-RNA substrate.