Comparative analysis of three laboratory based serological assays for SARS-CoV-2 in an Australian cohort.

Many unanswered questions remain regarding the role of SARS-CoV-2 serological assays in this unfolding COVID-19 pandemic. These include their utility for the diagnosis of acute SARS-CoV-2 infection, past infection or exposure, correlation with immunity and the effective duration of immunity. This study examined the performance of three laboratory based serological assays, EUROIMMUN Anti-SARS-CoV-2 IgA/IgG, MAGLUMI 2000 Plus 2019-nCov IgM/IgG and EDI Novel Coronavirus (COVID-19) IgM/IgG immunoassays. We evaluated 138 samples from a reference non-infected population and 71 samples from a cohort of 37 patients with SARS-CoV-2 confirmed positive by RT-PCR. The samples were collected at various intervals of 0-45 days post symptoms onset (PSO). Specificity and sensitivity of these assays was 60.9%/71.4% (IgA) and 94.2%/63.3% (IgG) for EUROIMMUN; 98.5%/18.4% (IgM) and 97.8%/53.1% (IgG) for MAGLUMI; and 94.9%/22.5% (IgM) and 93.5%/57.1% (IgG) for EDI, respectively. When samples collected ≥14 days PSO were considered, the sensitivities were 100.0 and 100.0%; 31.0 and 82.8%; 34.5 and 57.1%, respectively. Using estimated population prevalence of 0.1, 1, and 10%, the positive predictive value of all assays remained low. The EUROIMMUN Anti-SARS-CoV-2 IgA lacked specificity for acute diagnosis and all IgM assays offered poor diagnostic utility. Seroconversion can be delayed although all patients had seroconverted at 28 days in our cohort with the EUROIMMUN Anti-SARS-CoV-2 IgG. Despite this, with specificity of only 94% this assay would not be satisfactory for seroprevalence studies in the general Australian population given this is likely to be currently <1%.

Regulation of hepatic steroid receptors and enzymes by the 3beta-hydroxysteroid dehydrogenase inhibitor trilostane.

Therapies designed to treat hypercortisolism have usually sought to reduce circulating glucocorticoid concentrations, however the local tissue endocrine environment could be an alternative target. The 3beta-hydroxysteroid dehydrogenase Delta5-4 isomerase (3beta-HSD) inhibitor trilostane is of interest, since, although it is only moderately and transiently effective in reducing circulating steroid, it is remarkably effective in alleviating Cushing's symptoms in veterinary applications. To seek alternative modes of action, male Wistar rats were treated with trilostane. Although final circulating corticosteroid concentrations were unaffected, liver 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) transcription and translation was significantly increased, whereas 3beta-HSD was not affected either in liver or adrenal. Glucocorticoid receptor (GR) mRNA was down-regulated, and mineralocorticoid receptor (MR) up-regulated by trilostane treatment: no changes in 11beta-HSD1 mRNA were observed. Trilostane also had no direct effect on GR response element-mediated gene transcription. The results show that the tissue endocrine environment is affected by trilostane treatment in the absence of sustained changes in circulating corticosteroid. The combination of increased 11beta-HSD2 and reduced GR expression in target organs could be expected to ameliorate the effects of excess glucocorticoid, suggesting new therapeutic approaches.

ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen.

ShcA and Grb2 are crucial components in signalling by most tyrosine kinase-associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T-antigen (MT) of polyoma virus to address this question. The two known Grb2-binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA-interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane-bound ShcA requires only these two sequences and the ShcA-binding site in MT does not recruit other signalling molecules. Two standard Grb2-interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.

Relationship of numbing to alexithymia, apathy, and depression.

The present study assessed the relationship between numbing and three associated conditions of alexithymia, apathy, and depression, utilizing data collected on 353 Vietnam combat veterans diagnosed with Posttraumatic Stress Disorder from in- and out-patient settings and an outreach center at various Department of Veterans Affairs Medical centers. All subjects completed four self-report measures: the Glover Numbing Scale, the Beck Depression Inventory, the Apathy Evaluation Scale, and the Toronto Alexithymia Scale-20. The correlation matrix indicated that scores on the four measures were moderately to highly correlated. Principal components analysis with a varimax rotation indicated a five-factor solution that provided evidence for the factorial validity of each of the constructs assessed. Results of the factor analysis of items from the four measures were consistent with numbing being a separate and distinct construct from alexithymia, apathy, and depression. In general, results indicated that all constructs measured were separate and distinct from one another.

Association between src-kinases and the polyoma virus oncogene middle T-antigen requires PP2A and a specific sequence motif.

Polymoma virus encodes a potent oncogene, the middle T-antigen (MT), that induces cell transformation by copying the actions of tyrosine kinase associated growth factor receptors. A crucial component of MT transformation is its ability to bind and stimulate the activity of src-family kinases. However, the mechanism by which this is achieved remains unclear. Tyrosine phosphorylation of MT by src-kinases then provides binding sites for SH2 and PTB domain containing molecules in a paradigm of receptor action. We present evidence here that the MT/src complex contains equi-molar amounts of PP2A, and that phosphatase activity may be required for the interaction of MT with both PP2A and the src-family. PP2A, then, is a necessary component of the MT-src complex. We also show that two motifs in the 185 to 210 region of MT, each consisting of a basic area followed by a serine or threonine, are essential for interaction with src-kinases, but not PP2A. The spacing between the serine or threonine and the basic sequence also appears to be important. Substituting a cysteine residue in place of Thr203 in MT has no affect on the binding of pp60c-src, showing that these sites interact with src-kinases by a novel mechanism that does not require phosphorylation.

pp60c-src binding to polyomavirus middle T-antigen (MT) requires residues 185 to 210 of the MT sequence.

Interaction with the src family of tyrosine kinases is crucial to the transforming action of polyomavirus middle T-antigen (MT). Association with MT activates the tyrosine kinase activity of pp60(c-src) and, through subsequent MT phosphorylation, creates binding sites for signalling molecules whose stimulation culminates in cell transformation. Despite this importance, and many studies, little is known of the mechanisms by which pp60(c-src) binds to MT. We report here isolation of the first MT mutants that disrupt pp60(c-src) binding without affecting the interaction between MT and protein phosphatase 2A (PP2A). Through deletion analysis we established that interaction with pp60(c-src) requires the sequences between amino acids 185 and 210 of MT, but these residues have no effect on PP2A binding. Cells expressing these mutants showed few altered properties, indicating that the PP2A-MT interaction alone has little influence on cell phenotype. Subcellular location of these mutant MT molecules was indistinguishable by immunofluorescence analysis from that of wild-type MT but was altered markedly on loss of PP2A binding. This suggests a possible role for PP2A in specifying subcellular distribution.

Numbing scale scores in female psychiatric inpatients diagnosed with self-injurious behavior, dissociative identity disorder, and major depression.

Female inpatients engaged in self-injurious behavior (SIB) and females diagnosed with Dissociative Identify Disorder (DID) scored higher on the Glover Numbing Scale (GNS) than female inpatients diagnosed with Major Depressive Disorder (MDD). The DID sample showed a multi-modal distribution of scores, and the MDD sample showed a bimodal distribution with a significant difference between the means of the two subgroups. An additional subsample of outpatient males diagnosed with MDD also evidenced a bimodal distribution of scores with a similar spread between the two means. Scores on the Beck Depression Inventory did not discriminate the latter two subgroups.

Effect of instruction to sing on stuttering frequency at normal and fast rates.

Singing as a fluency-enhancing mechanism is well-established. The fluency derived by singing has been attributed to a reduced speech rate, memorized material, semantically reduced content, and an imposed rhythm. In this study, we attempted to address each of these explanations. 12 participants who stuttered were instructed to read or sing each of four different passages under the following conditions: reading at a normal rate, reading at a fast rate, singing at a normal rare, and singing at a fast rate. Participants exhibited a statistically significant increase in disfluencies while reading, i.e., participants displayed a 75% reduction in disfluency in the singing condition relative to the reading condition. There was no difference in stuttering frequency with rate conditions. Current findings suggest that stutterers are capable of internally generating fluent speech production by imposing idiosyncratic melodic structures or some derivation of melody when asked simply to sing. There is no claim that these participants were singing, as skills and capabilities varied tremendously, only that participants achieved dramatic enhancement of fluency after they were just asked to sing. Thus, the only intervening variable was the instruction to sing, which suggests the attempt to follow the instruction, no matter how futile, generated fluent speech. Since fluency was maintained in both the normal and fast rates of production, alternate central mechanisms must be held accountable for these findings.

Vulnerability Scale scores in female inpatients diagnosed with self-injurious behaviour, dissociative identity disorder, and major depression.

For three groups of predominantly female inpatients scores on the Glover Vulnerability Scale were compared with each other and with those of a control sample. The four groups included individuals who were diagnosed with self-injurious behavior, females diagnosed with Dissociative Identity Disorder, females whose predominant diagnosis was Major Depressive Disorder, and female controls. Females with a trauma-related diagnosis (Groups 1 and 2) scored higher on the Glover Vulnerability Scale than Group 3. History of exposure to trauma or childhood abuse most clearly discriminated higher scores within the control sample. The findings highlighted the close relationship between histories of exposure to trauma and high scores on this scale. Additional analysis of these scores and those on the Glover Numbing Scale showed a close association between feelings of vulnerability and the numbing response.

Selection of activating mutations of c-fms in FDC-P1 cells.

FDC-P1 haemopoietic cells were used to select mutations of c-fms that constitutively activate the receptor for macrophage-colony stimulating factor (M-CSF or CSF-1). One mutation changed Ser 929 to Gly within a Ser/Gly rich region of the C-terminal tail and a second changed a nearby, highly conserved Leu 926 for Pro. A third mutation (D802V) changed Asp 802 to Val within the alpha L12/beta 9 region of the tyrosine kinase domain, so supporting the crystallographic evidence that this region triggers kinase activation. A c-kit mutation exactly equivalent to D802V was previously identified in a leukamic cell line and was demonstrated here to be transforming. Surprisingly, although D802V potently transformed FDC-P1 cells, it could not induce Rat-2 fibroblast foci, even in the presence of M-CSF. It is suggested that the accelerated receptor degradation induced by D802V may account for its cell specific effect.

Vulnerability Scale: a preliminary report of psychometric properties.

This work describes assessment of the psychometric properties of a self-report instrument, the Glover Vulnerability Scale. This scale was administered to a total of 11 groups (N = 695). Six of the groups were Vietnam combat veterans diagnosed as having Posttraumatic Stress Disorder (n = 531). The estimate of internal consistency was .88; the test-retest correlation over 4 wk. was .81. Convergent and discriminant validations were satisfactory based on the pattern of the scale's correlations with relevant MMPI subscales and demographic data. Scale scores also discriminated levels of functioning within the population diagnosed with Posttraumatic Stress Disorder and discriminated veterans diagnosed with Posttraumatic Stress Disorder from patients with major depressive disorder and anxiety disorder. Principal component factor analysis gave a 4-factor solution: social comfort, vulnerability, paranoia, and family trust. Over-all, the findings strongly support the clinical application of the Vulnerability Scale.

Purification of three pectin esterases from ripe peach fruit.

Three isoforms of pectin esterase (PE1-PE3) (pectin pectyl-hydrolase, EC 3.1.1.11.) were purified to homogeneity from ripe peach fruit (Prunus persica cv. Coronet). The three enzymes were basic proteins of M(r) 34,000 as determined by denaturing polyacrylamide gel electrophoresis but were separated by FPLC cation-exchange chromatography. The proteins were N-terminally blocked but amino acid sequences were obtained for peptides released from two of the three isoforms. The sequences revealed a threonine/lysine substitution in a comparison between isoform PE2/isoform PE3, and there were regions of sequence similarity with other plant pectin esterases. The proteins did not bind to concanavalin A and were not stained by the periodate-Schiff reagent suggesting a low or zero level of glycosylation. Polyclonal antisera to isoform PE3 also bound to isoforms PE1 and PE2. The study provides the enzyme protein sequence and immunological basis for an evaluation of the role of pectin esterases in normal and abnormal ripening of peach fruit.

Synergy between SCF or M-CSF with IL-3 or GM-CSF in FDC-P1 cells: a sensitive assay of transforming mutations of c-fms.

Stem cell factor (SCF) was found to stimulate the growth of the haemopoietic cell line FDC-P1 in synergy with either interleukin 3 (IL-3) or granulocyte-macrophage-colony stimulating factor (GM-CSF). Similarly, macrophage colony-stimulating factor (M-CSF) was shown to synergize with IL-3 or GM-CSF, following the infection of FDC-P1 cells with a recombinant retrovirus which encoded the receptor for M-CSF (M-CSFr). These results raise the possibility that signal transduction pathways which are controlled by SCF in FDC-P1 cells, can be activated by M-CSF if its receptor is illicitly expressed. FDC-P1 cells that expressed the M-CSFr were responsive to as little as 100 U/ml of M-CSF when added in combination with IL-3 or GM-CSF. This sensitive assay was used to demonstrate that transforming deletions of the C-terminal tail of the M-CSFr and two-point mutations within the same region that converted tyrosine 969 to either phenylalanine or to cysteine, allowed the mutant M-CSF receptors to synergize with IL-3 or GM-CSF in the absence of M-CSF. These mutations were found to be more evidently transforming in FDC-P1 cells than in Rat-2 fibroblasts. The possible relevance of these results to leukaemia and to gynaecological malignancies is discussed.

The Numbing Scale: psychometric properties, a preliminary report.

This study explored the psychometric properties of a new self-report instrument, The Glover Numbing Scale. The scale measures a variety of behaviors reported by individuals experiencing an inability to access feelings other than hostility and rage. The scale was administered to inpatient Vietnam combat veterans with post-traumatic stress disorder (PTSD, n = 323), PTSD diagnosed Veterans Affairs (VA) outpatient and veterans' outreach center veterans (n = 208), two Vietnam veteran noncombat groups (n = 45), two psychiatric noncombat controls (anxiety disorder, n = 40; major depressive disorder, n = 31), and a nonpsychiatric never-in-Vietnam veteran control group (n = 48). Reliability information suggested that the scale was internally consistent with good test-retest correlations. Convergent and discriminant validations were assessed based on the pattern of the scale's correlations with relevant Minnesota Multiphasic Personality Inventory (MMPI) scales. The contrasting pattern of correlations provided by a subgroup of veterans who reported always feeling dead or shut down was highlighted. Principal component analysis resulted in a five factor solution that provided evidence for the scale's factorial validity. Numbing Scale scores discriminated levels of psychopathology within the veteran population. PTSD outreach center veterans' sum scores and item scores on the Numbing Scale were more similar to responses of anxiety disorder patients than to major depressive disorder patients. Overall, the findings strongly support the clinical application of the Numbing Scale.

A preliminary trial of nalmefene for the treatment of emotional numbing in combat veterans with post-traumatic stress disorder.

Eighteen chronic post-traumatic stress disorder (PTSD+) combat veterans were administered nalmefene, a non-FDA approved oral opiate antagonist, based on the hypothesis that emotional numbing is an opiate-mediated phenomenon. Eight veterans showed a favorable response with a marked decrease of emotional numbing and other symptoms of PTSD, including startle response, nightmares, flashbacks, intrusive thoughts, rage and vulnerability. Veterans who responded favorably to the drug demonstrated a waxing and a waning of positive and negative PTSD symptoms with a general improvement in all these symptoms at higher nalmefene dosages. Case descriptions highlight some of the many changes veterans experienced on nalmefene dosage increases.

Performance of amblyopic children on printed contrast sensitivity test charts.

Forty consecutive amblyopic patients between the ages of 3 and 12 years attending the Orthoptic Clinic for assessment and treatment were tested on the AO Contrast Sensitivity System [Arden grating test (AGT)] and the VISTECH Contrast Sensitivity Charts. Recordings were unsuccessful in more than 50% of these patients on both test systems. Of those who could successfully complete the AGT, false negative results were noted in 62.5% of the amblyopic eyes and false positive results were found in 25% of the normal fellow eyes. On the near VISTECH chart, all normal and amblyopic eyes produced results that were outside the specified limit of normality. On the distance VISTECH chart, 25% of the normal fellow eyes gave false positive results and 25% of the amblyopic eyes gave false negative results. From these findings, it is doubtful whether either test system has a role in the routine clinical assessment of amblyopia.