Recommendations for the classification and nomenclature of the DNA-β satellites of begomoviruses

The symptom-modulating, single-stranded DNA satellites (known as DNA-β) associated with begomoviruses (family Geminiviridae) have proven to be widespread and important components of a large number of plant diseases across the Old World. Since they were first identified in 2000, over 260 full-length sequences (∼1,360 nucleotides) have been deposited with databases, and this number increases daily. This has highlighted the need for a standardised, concise and unambiguous nomenclature for these components, as well as a meaningful and robust classification system. Pairwise comparisons of all available full-length DNA-β sequences indicate that the minimum numbers of pairs occur at a sequence identity of 78%, which we propose as the species demarcation threshold for a distinct DNA-β. This threshold value divides the presently known DNA-β sequences into 51 distinct satellite species. In addition, we propose a naming convention for the satellites that is based upon the system already in use for geminiviruses. This maintains, whenever possible, the association with the helper begomovirus, the disease symptoms and the host plant and provides a logical and consistent system for referring to already recognised and newly identified satellites.     

Disconnection of man and the soil: reason for the asthma and atopy epidemic?

Intense search has been going on to find factors responsible for the asthma and atopy epidemic in Western societies. Attention has increasingly been devoted to environmental saprophytes, which, in addition to gut commensals, might be the major players in the development and fine tuning of immunologic homeostasis. This review outlines current evidence for the role of environmental saprophytes in the development of atopic disease and considers the consequences of urbanization in reducing contacts with soil microorganisms. The major microbial components that have been shown to possess immunomodulatory capacity and their respective Toll-like receptors are also discussed, as are the possible mechanisms underlying the ability of saprophytes to confer protection against atopic disease.     

Evaluation of the CELL-DYN® 3500 haematology instrument for the analysis of the mouse and rat blood

The objective of this study was to evaluate the performance of the CELL-DYN^® 3500 for rat and mouse blood analysis in a routine environment. The WBC (white blood cells), RBC (red blood cells), PLT (platelets) counts and the WBC differential were determined. In addition, the following aspects were studied: within-run precision, day-to-day precision, biasfree paired difference precision; extended ranges of linearity for RBC, HCT (haematocrit), WBC, PLT; carry-over, the fffect of blood ageing, cell stability with different anticoagulants; and the normal ranges, the out of range flagging and some typical pathology cases. The CELL-DYN^® 3500 is a multiparameter flow cytometer which counts and differentiates WBC, based on the principle of multi-angle polarised light scatter separation. RBC and PLT are determined by the impedance method. The WBC count is evaluated by both, optical and impedance methods. Reference methods used were according to the ICSH recommendations on blood cell analysis, including manual counts of WBC and platelets, a centrifugal microhaematocrit method and a haemoglobin measurement by spectrophotometry using the WHO haemoglobin standard. All cell counts were compared with the results obtained by our routine blood cell analyser (Contraves AL820), and the WBC differential was compared with the manual microscopic differentiation of the 400 WBC (200 cells differentiated by two technicians). The following coefficients of variation were obtained: within-run precision was 1.2% and 2.7% for WBC; 1.0% and 1.0% for RBC; 1.3% and 0.9% for haematocrit; 2.1% and 2.7% for platelets (rats and mice respectively). Day-to-day precision was performed using human trilevel control blood, and the CVs were found to be <1.7% for WBC, <1.4% for RBC, <1.2% for haemoglobin and <6.3% for platelets. The following ranges of measurement were found to be linear in the rat: WBC: 0.10–20.20×10³/μl; RBC: 0.016–14.3×10⁶/μl; haemoglobin: 0.08–26.8 g/dl; haematocrit: 5.0%–77%; platelets: 14.0–1670.0×10³/μl. Equal ranges were observed for mouse blood. Carry-over in rat blood was found to be 0.12% for WBC, 0.05% for RBC, 0.15% for haemoglobin and 0.46% for platelets. In mice, similar carry-over results were obtained. The correlation coefficients (Pearson, correlation coefficient) between the CELL-DYN^® 3500 and Contraves AL 820 using linear regression analysis were as follows: 0.988 and 0.997 for WBC; 0.986 and 0.920 for RBC; 0.995 and 0.984 for haemoglobin; 0.958 and 0.85 for haematocrit; 0.958 and 0.963 for platelets, for rats and mice, respectively. Correlation coefficients between the CELL-DYN^® 3500 and the manual differential of NEU (neutrophils) and LYM (lymphocytes) were higher than 0.8 in rats and higher than 0.9 in mice. Due to the relatively low absolute counts of MONO (monocytes), EOS (eosinophils) and BASO (basophils), only moderate correlation of methods was found. The CELL-DYN^® 3500 was judged to be reliable, accurate and easy-to-use for counting and identifying normal and most of the pathological blood specimens obtained from mice and rats. By using the CELL-DYN^® 3500, the time for blood sample analysis can be shortened significantly and provides extensive opportunities to characterise pathological samples.     

Influence of storage on the quality of conventional CT and µCT-imaging for the middle and inner cat ear

The aim of this study was to analyze whether different fixation methods such as freezing or formaldehyde storage for different periods of time have an influence on the recognition of anatomical relevant structures in the middle and inner ear of the cat with conventional computed tomography (cCT) and micro-computed tomography (µCT). Besides, effects of freeze–thaw cycles on determined structures of the ear were investigated by means of histological slices. Three veterinarians with different radiologic expertise evaluated the scans of 30 dissected cat ears anonymously and scored predefined structures in a five-point scale with reference to visually sharp reproducibility and perfect image quality. The total scores of the different fixation groups as well as the ears within a group were compared with each other. Furthermore, an intra-reader examination including an evaluation of the identifiability of specified structures was performed for both imaging methods. cCT as well as µCT-scans have a very low variation coefficient of 1.6% and 2.3%, respectively. The results for the alterations between the different fixation methods show that the changes for cCT-scans are negligible, as the percentage alteration compared to fresh samples ranges in a very small interval with values from 1.0% better to 1.2% worse. µCT-scans are more influenced by the fixation method with a range from 1.3% better to 6.9% worse values. The scans mostly deteriorated after two freeze–thaw cycles (1.8% worse) and after storing the samples for 1 (2.4% worse), respectively, and 3 weeks in formaldehyde (6.9% worse).

     

Evaluation of the combination of the NucliSENS easyMAG® and the EasyQ® applications for the detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory tract specimens

Mycoplasma pneumoniae and Chlamydia pneumoniae are respiratory tract pathogens frequently involved in community-acquired pneumonia, but are fastidious microorganisms. Their direct detection mainly requires molecular amplification techniques. A nucleic acid extraction system, NucliSENS easyMAG®, and a real-time nucleic acid sequence-based amplification (NASBA) technique, NucliSENS EasyQ®, were recently developed by bioMérieux to detect both bacteria. The aim of our study was to compare the easyMAG/EasyQ combination with our in-house combination, MagNA Pure extraction (Roche) and real-time polymerase chain reaction (PCR), to detect both bacteria in respiratory tract specimens. The analytical specificities of both combinations were similar. A higher analytical sensitivity was found for C. pneumoniae using the easyMAG/EasyQ combination, since the easyMAG/EasyQ system detected nucleic acid extracts 10⁶ times more diluted than the in-house combination. Both combinations were equivalent when detecting M. pneumoniae in positive respiratory tract samples. Finally, the easyMAG/EasyQ combination is a potential useful tool for the detection of both bacteria regarding sensitivity, specificity, monitoring, and standardization of the procedure.     

Canalicular Adenoma—Search for the Cell of Origin: Ultrastructural and Immunohistochemical Analysis of 7 Cases and Review of the Literature

Abstract

Canalicular adenoma (CA) is a rare, benign epithelial neoplasm of the salivary glands. Historically considered to be a variant of basal cell adenoma, this “monomorphic” adenoma has a distinct clinical, morphologic, and immunohistochemical profile. The putative cell of origin remains a topic of debate. A combination of morphology, immunohistochemistry, and ultrastructural analyses have been employed to determine histogenesis, but the interpretations of these studies have implicated multiple different cell types along the salivary gland duct as the cell of origin. The authors sought to further characterize CA using electron microscopy, immunohistochemistry, and special and immuno-stains on 7 cases. Their morphologic, immunohistochemical, and ultrastructural findings support a cell of origin demonstrating features of both the intercalated duct cells and the striated duct luminal epithelial cells.     

Early Reconstitution of NK and γδ T Cells and Its Implication for the Design of Post-Transplant Immunotherapy

Relapse is the most frequent cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Natural killer (NK) cells and γδ T cells reconstitute early after allo-HSCT, contribute to tumor immunosurveillance via major histocompatibility complex–independent mechanisms and do not induce graft-versus-host disease. Here we performed a quantitative and qualitative analysis of the NK and γδ T cell repertoire in healthy individuals, recipients of HLA-matched sibling or unrelated donor allo-HSCT (MSD/MUD-HSCT) and umbilical cord blood-HSCT (UCB-HSCT). NK cells are present at high frequencies in all allo-HSCT recipients. Immune reconstitution (IR) of vδ2⁺?cells depended on stem cell source. In MSD/MUD-HSCT recipients, vδ2⁺?comprise up to 8% of the total lymphocyte pool, whereas vδ2⁺?T cells are barely detectable in UCB-HSCT recipients. Vδ1⁺?IR was driven by CMV reactivation and was comparable between MSD/MUD-HSCT and UCB-HSCT. Strategies to augment NK cell mediated tumor responses, similar to IL-15 and antibodies, also induced vδ2⁺?T cell responses against a variety of different tumor targets. Vδ1⁺?γδ T cells were induced less by these same stimuli. We also identified elevated expression of the checkpoint inhibitory molecule TIGIT (T cell Ig and ITIM domain), which is also observed on tumor-infiltrating lymphocytes and epidermal γδ T cells. Collectively, these data show multiple strategies that can result in a synergized NK and γδ T cell antitumor response. In the light of recent developments of low-toxicity allo-HSCT platforms, these interventions may contribute to the prevention of early relapse.     

Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases

Extended spectrum β-lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. Threat posed by antibiotic resistance because of ESBLs is more serious as a number of technical problems are associated with the detection of these enzymes. Although a number of detection methods have been designed for ESBLs, every method has its own benefits and shortcomings as well. In earlier days, isoelectric focusing (IEF) was used as the gold standard for ESBL detection. This study was undertaken to compare IEF with polymerase chain reaction, a method which has been extensively used for ESBL detection these days.     

What is the current status of chondroitin sulfate and glucosamine for the treatment of knee osteoarthritis?

Chondroitin sulfate and glucosamine sulfate exert beneficial effects on the metabolism of in vitro models of cells derived from synovial joints: chondrocytes, synoviocytes and cells from subchondral bone, all of which are involved in osteoarthritis (OA). They increase type II collagen and proteoglycan synthesis in human articular chondrocytes and are able to reduce the production of some pro-inflammatory mediators and proteases, to reduce the cellular death process, and improve the anabolic/catabolic balance of the extracellular cartilage matrix (ECM). Clinical trials have reported a beneficial effect of chondroitin sulfate and glucosamine sulfate on pain and function. The structure-modifying effects of these compounds have been reported and analyzed in recent meta-analyses. The results for knee OA demonstrate a small but significant reduction in the rate of joint space narrowing. Chondroitin sulfate and glucosamine sulphate are recommended by several guidelines from international societies for the management of knee and hip OA, while others do not recommend these products or recommend only under condition. This comprehensive review clarifies the role of these compounds in the therapeutic arsenal for patients with knee OA.     

Characteristics of Radioimmunoassays for the α- and β-Subunits of Human Luteinizing Hormone

Abstract

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-α and hLH-β) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-α and anti-hLH-β antisera were calculated at 157 ± 8.4 nM^?1 and 109 ± 7.4 nM^?1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-α RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-α, was equipotent with the hLH-α standard in this assay. In the homologous hLH-β RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-β, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-α and hLH-β while the hFSH RIA can be used without correction for the presence of the three other components.     

Evaluation of the CELL-DYN? 3500 haematology instrument for the analysis of the mouse and rat blood

The objective of this study was to evaluate the performance of the CELL-DYN^? 3500 for rat and mouse blood analysis in a routine environment. The WBC (white blood cells), RBC (red blood cells), PLT (platelets) counts and the WBC differential were determined. In addition, the following aspects were studied: within-run precision, day-to-day precision, biasfree paired difference precision; extended ranges of linearity for RBC, HCT (haematocrit), WBC, PLT; carry-over, the fffect of blood ageing, cell stability with different anticoagulants; and the normal ranges, the out of range flagging and some typical pathology cases. The CELL-DYN^? 3500 is a multiparameter flow cytometer which counts and differentiates WBC, based on the principle of multi-angle polarised light scatter separation. RBC and PLT are determined by the impedance method. The WBC count is evaluated by both, optical and impedance methods. Reference methods used were according to the ICSH recommendations on blood cell analysis, including manual counts of WBC and platelets, a centrifugal microhaematocrit method and a haemoglobin measurement by spectrophotometry using the WHO haemoglobin standard. All cell counts were compared with the results obtained by our routine blood cell analyser (Contraves AL820), and the WBC differential was compared with the manual microscopic differentiation of the 400 WBC (200 cells differentiated by two technicians). The following coefficients of variation were obtained: within-run precision was 1.2% and 2.7% for WBC; 1.0% and 1.0% for RBC; 1.3% and 0.9% for haematocrit; 2.1% and 2.7% for platelets (rats and mice respectively). Day-to-day precision was performed using human trilevel control blood, and the CVs were found to be <1.7% for WBC, <1.4% for RBC, <1.2% for haemoglobin and <6.3% for platelets. The following ranges of measurement were found to be linear in the rat: WBC: 0.10–20.20×10³/μl; RBC: 0.016–14.3×10⁶/μl; haemoglobin: 0.08–26.8 g/dl; haematocrit: 5.0%–77%; platelets: 14.0–1670.0×10³/μl. Equal ranges were observed for mouse blood. Carry-over in rat blood was found to be 0.12% for WBC, 0.05% for RBC, 0.15% for haemoglobin and 0.46% for platelets. In mice, similar carry-over results were obtained. The correlation coefficients (Pearson, correlation coefficient) between the CELL-DYN^? 3500 and Contraves AL 820 using linear regression analysis were as follows: 0.988 and 0.997 for WBC; 0.986 and 0.920 for RBC; 0.995 and 0.984 for haemoglobin; 0.958 and 0.85 for haematocrit; 0.958 and 0.963 for platelets, for rats and mice, respectively. Correlation coefficients between the CELL-DYN^? 3500 and the manual differential of NEU (neutrophils) and LYM (lymphocytes) were higher than 0.8 in rats and higher than 0.9 in mice. Due to the relatively low absolute counts of MONO (monocytes), EOS (eosinophils) and BASO (basophils), only moderate correlation of methods was found. The CELL-DYN^? 3500 was judged to be reliable, accurate and easy-to-use for counting and identifying normal and most of the pathological blood specimens obtained from mice and rats. By using the CELL-DYN^? 3500, the time for blood sample analysis can be shortened significantly and provides extensive opportunities to characterise pathological samples.     

Is the integration of heard and seen speech mandatory for infants?

For adults and children, speech perception can be significantly influenced by watching a speaker's mouth movements. While recent reports suggest that infants may be able to integrate heard and seen speech, the current research demonstrates that integration is neither as strong or consistent in infants as it is in adults. Three habituation experiments were conducted. In Experiment 1, female (but not male) infants showed evidence of an adult pattern of integration following habituation with an audiovisual /bi/ and testing with audio /bi/-visual /vi/ (perceived as /vi/ by adults). The interpretation of integration was supported, but only in part, by Experiment 2. In Experiment 3, infants were habituated to a mismatched audio /bi/-visual /vi/ combination and tested on concordant /bi/-/bi/ versus /vi/-/vi/ displays. Here, only male infants showed evidence of integration. These results suggest that an initial mechanism supports integration, but that integration is not mandatory for young infants.     

Conserved nucleotides in the terminus of the 3′ UTR region are important for the replication and infectivity of porcine reproductive and respiratory syndrome virus

The 3′ untranslated region (3′ UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3′ UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3′ UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3′ UTR region. Our findings indicated that the conservation of the primary sequence of the 3′ terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5′-¹⁵⁵¹⁷AAUU¹⁵⁵²⁰-3′) at the 3′ proximal end of the 3′ UTR and the dinucleotide 5′-AU-3′ exerted an important regulatory effect on viral viability. Of the five 3′-terminal nucleotides of the 3′ UTR (5′-¹⁵⁵⁰³AACCA¹⁵⁵⁰⁷-3′), at least three, including the last dinucleotide (5′-CA-3′), were essential for maintaining viral infectivity. Taken together, the 3′-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex.     

Significance of the antigenic epitopes in heterogenous nuclear ribonucleoproteins-Ⅰ for the diagnosis and prognosis of systematic sclerosis

To assess the presence of autoantibodies against epitopes of heterogenous nuclear ribonucleoprotein-Ⅰ(hnRNP-Ⅰ) in systematic sclerosis (SSc) and to analyze their clinical significance, polypeptides of hnRNP-Ⅰwere designed by biological technical software and analyzed with both the Wonderful Biology Information System and DNA Star-Protean Analysis Software at the same time. In these ways, two polypeptides of hnRNP-Ⅰwere obtained based on their amino acid sequences, folding features, hydrophilic, curl style, dough kneading sensation and the possibility on the surface of proteins. They are named as hnRNP-Ⅰ-1 ( NVKYNNDKSRDYTRPDLPSGDSQPSLDQT, 264-292 aa) and hnRNP-Ⅰ-2 (QLP4REGQEDQGLTKDYGNSOL, 441-461 aa), simply designated asⅠ-1 andⅠ-2. The autoantibodies against hnRNPs were detected by means of ELISA using the synthetic epitopes polypeptides as antigen. It was found that the positive rate of detection for anti-Ⅰ-1 and anti-Ⅰ-2 autoantibodies were rather higher in SSc patients than that in other CTDs and the sensitivities and specificities of the testing with ELJSA for anti-Ⅰ-1 and anti-Ⅰ-2 antibodies in SSc patients were 47. 62%/93. 43% and 38. 1%/ 91.08% , without any significant difference between these two groups of testings. Also, there was no significant difference in the clinical features and laboratory findings, such as age, involvements in digestive and respiratory tracts and erythrocyte sedimentation rate etc., between the anti-Ⅰ-1-positive and -negative groups in SSc patients. However, the hnRNP-Ⅰ-autoantibody-positive group of patients had obviously shorter duration of disease course compared with that of the autoantibody-negative group. Anti-Ⅰ-1 and anti-Ⅰ-2 autoantibodies also had no association with antinuclear antibody, anti-Scl70 and anti-centromere antibody (ACA) in SSc patients. So, it is apparent that the autoantibodies related with SSc may act through different pathways in the pathogenesis of SSc, and the hnRNP-Ⅰautoantibody is a new type antibody occuring during the early stage of disease and appearing to have diagnostic and prognostic significances .     

Targeting the junction of CɛmX and ɛ-migis for the specific depletion of mIgE-expressing B cells

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the C?mX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ?-migis. In the short isoform the C?mX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ?-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ?-migis and (2) the junction of C?mX and ?-migis (C?mX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ?-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ?-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the C?mX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which C?mX is absent. These results suggest that C?mX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation.