Evaluating the Ability to ID (COVID-19) NOW: a Large Real-World Prospective Evaluation of the Abbott ID NOW COVID-19 Assay.

The Abbott ID NOW COVID-19 assay is a rapid point-of-care molecular test for SARS-CoV-2 detection. In theory, it has the potential to decrease turnaround times (TATs) and rapidly facilitate patient flow and triage. Reports for its performance have been mixed, likely due to variations in patient cohorts, preanalytical considerations, and study design. We prospectively evaluated the ID NOW performance against reference reverse transcriptase PCR (RT-PCR) tests, using dual swabs. Patients presented at a large multisite academic hospital with the highest volumes of COVID-19 admissions in Canada. From 1,968 valid swabs, 186 were true positive, 1,760 were true negative, 21 were false negatives, and 1 was false positive. At 10.5% positivity rate, the positive and negative predictive values were 99.5% and 98.8%, respectively. This led to a modest increase in the pretest probability in this cohort of individuals presenting <7 days of symptom onset. The mean times from collection to laboratory receipt and receipt to reporting were 31 and 23 min, respectively. This reduced TAT observed in our study may assist with triage of admitted patients and breaking the chain of transmission through immediate notification of status. We also observed how test performance changed with prevalence, and thus, how the test is used to "rule in" or "rule out" disease must be considered. Although the ID NOW is regarded as a rapid test, it is not high throughput and requires rapid transportation times (<1 h) that may not be plausible in large centers. The utility of this test should be considered with the observed TAT and interpreted in the context of limitations discussed. IMPORTANCE Rapid testing for COVID-19 has been recognized as one potentially important measure in managing the pandemic. However, these rapid tests vary grossly in their performance and their applicability. There have been many studies evaluating the performance of rapid tests for SARS-CoV-2 detection. However, they are frequently not prospective, and patients are not simultaneously swabbed to compare the reference standard RT-PCR. Previous ID NOW study findings are mixed, which may be due to various factors, including patient, epidemiological, and preanalytical considerations. It is critical to consider how the pretest and posttest probabilities and epidemiological factors may affect the performance as the community prevalence of disease fluctuates during this highly dynamic pandemic. We consider how the ID NOW may be utilized in different settings, with considerations of public health and infection control and prevention risk tolerance.

Transcriptional repression of type I procollagen genes during adipocyte differentiation.

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.

Antagonistic regulation of protein kinase C-induced stimulation of fibronectin synthesis by cyclic AMP in human lung fibroblasts.

We studied the interaction between cAMP and protein kinase C (PKC) signaling pathways in the regulation of fibronectin synthesis in human lung fibroblasts. Phorbol myristate acetate (PMA), a PKC activator, stimulated fibronectin synthesis and its mRNA expression in both normal and transformed human lung fibroblasts (WI-38 and WI-38 VA13, respectively). On the other hand, dibutyryl cAMP (Bt2cAMP), a cAMP analogue, did not alter fibronectin synthesis in both cell lines. The combined treatment of Bt2cAMP with PMA, however, suppressed the PMA-induced stimulation of fibronectin synthesis and mRNA expression in these cells. This study shows that cAMP pathway antagonizes PKC pathway in regulating fibronectin synthesis in human lung fibroblasts and provides an example of antagonistic interaction between cAMP and PKC signaling pathways.

Okadaic acid increases fibronectin synthesis in MC3T3-E1 cells.

Okadaic acid, a potent specific inhibitor of serine/threonine protein phosphatases type 1 and 2A, affects several gene expressions in various cells. To determine whether okadaic acid affects the expression of fibronectin in MC3T3-E1 cells, we measured mRNA level and synthesis of fibronectin by Northern blot hybridization and immunoprecipitation methods, respectively. Okadaic acid (10-50 ng/ml) increased both mRNA level and synthesis of fibronectin in a dose-dependent manner. The increase of fibronectin mRNA by okadaic acid was strongly attenuated by the inhibition of new protein synthesis. The results indicate that okadaic acid, inhibitor of protein phosphatases, increases fibronectin synthesis in MC3T3-E1 cells.

Stimulation of fibronectin synthesis through the protein kinase C signalling pathway in normal and transformed human lung fibroblasts.

We examined the role of the protein kinase C (PKC) signaling pathway in the stimulation of fibronectin synthesis in both normal and transformed human lung fibroblasts. Phorbol myristate acetate (PMA), a potent PKC activator, stimulated fibronectin synthesis in both normal and transformed fibroblasts in a time and dose dependent fashion. Down-regulation of PKC by prior exposure of cells to a high concentration of PMA blocked the increase in fibronectin synthesis and mRNA levels induced by PMA. Bisindolylmaleimide, a specific inhibitor of PKC, also abolished the PMA-induced fibronectin synthesis. 4 alpha-phorbol didecanoate, an inactive phorbol ester, failed to affect fibronectin synthesis. These data suggest that PMA stimulates fibronectin synthesis and gene expression through the PKC signaling pathway in both normal and transformed human lung fibroblasts.

Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells.

Bone formation involves several tightly regulated gene expression patterns of bone-related proteins. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone-related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4-10), bone matrix formation/maturation (days 10-16), and mineralization stages (days 16-30). During the proliferation period (days 4-10), the expression of cell-cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10-16), type I collagen expression and biosynthesis, fibronectin, TGF-beta 1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF-beta 1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16-30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone-related proteins are temporally regulated during the MC3T3-E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone-related proteins in relation to the different stages during the osteoblast differentiation.

Three classes of mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex.

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.

Okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.

Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.

Studies on the structure of the mouse CBF-A gene and properties of a truncated CBF-A isoform generated from an alternatively spliced RNA.

CCAAT-binding factor (CBF), a heteromeric transcription factor that binds to sequences containing a CCAAT motif, is composed of three subunits, A, B and C, which are all required for DNA binding. The mouse CBF-A gene contains seven coding exons, which span 12 kb. Evidence is also presented for an additional 5' untranslated exon. The 90-amino-acid (aa) segment of CBF-A, which shows a high degree of sequence identity with the yeast transcription factor, HAP3, is split into exons 3 and 4. An alternatively spliced RNA that lacks exon 3 was identified by polymerase chain reaction. Although removal of exon 3 interrupts the CBF-A reading frame, a potential start codon at the 3' end of exon 2 is in the same reading frame as the reading frame encoding CBF-A in exons 4 to 7. A CBF-A polypeptide of the predicted 17-kDa, size, was indeed identified after in vitro transcription and translation of the DNA complementary to RNA (cDNA) corresponding to the alternatively spliced CBF-A mRNA. In contrast to full-length CBF-A, this truncated CBFA did not bind to a DNA sequence containing the CCAAT motif in the presence of the other two components of CBF. This result indicates that the segment corresponding to the exons missing in the truncated isoform of CBF-A is essential for the binding of CBF to DNA.

Selective inhibition of collagen synthesis by okadaic acid in cultured human fibroblasts.

To determine whether protein phosphatases can affect collagen synthesis, we examined the effect of okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, on collagen synthesis. Okadaic acid significantly decreased the [3H]proline incorporation into the collagenase-digestible protein and the percent collagen synthesis. These effects were synergistic with phorbol myristate acetate (PMA). The time course study showed that okadaic acid inhibited collagen synthesis after a 12 h treatment while PMA inhibited at 3 h. Down-regulation of protein kinase C by chronic treatment with PMA did not abrogate the okadaic acid-dependent inhibition. These results provide evidence for the involvement of protein phosphatases in the regulation of collagen synthesis.