13 research outputs found
Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints
A highly specific and sensitive serological assay detects SARS‑CoV‑2 antibody levels in COVID‑19 patients that correlate with neutralization
Objective
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti SARS-CoV-2 antibody levels.
Methods
In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated.
Results
The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum.
Conclusions
With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a
preferred tool to assess thresholds of protective immunity after infection and vaccination
Earliest Jurassic Patellogastropod, Vetigastropod, and Neritimorph Gastropods from Luxembourg with Considerations on the Triassic—Jurassic Faunal Turnover
Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints
Purpose To understand cell cycle controls in the 8-Cell human
blastomere.
Methods Data from whole human genome (43,377 elements) microarray
analyses of RNAs from normal 8-Cell human embryos were compiled with
published microarrays of RNAs from human fibroblasts, before and after
induced pluripotency, and embryonic stem cells. A sub database of 3,803
genes identified by high throughput RNA knock-down studies, plus genes
that oscillate in human cells, was analyzed.
Results Thirty-five genes over-detected at least 7-fold specifically on
the 8-Cell arrays were enriched for cell cycle drivers and for proteins
that stabilize chromosome cohesion and spindle attachment and limit DNA
and centrosome replication to once per cycle.
Conclusions These results indicate that 8-cell human blastomere cleavage
is guided by cyclic over-expression of key proteins, rather than
canonical checkpoints, leading to rapidly increasing gene copy number
and a susceptibility to chromosome and cytokinesis mishaps, well-noted
characteristics of preimplantation human embryos
Evidence that human blastomere cleavage is under unique cell cycle control
To understand the molecular pathways that control early human embryo
development.
Improved methods of linear amplification of mRNAs and whole human genome
microarray analyses were utilized to characterize gene expression in
normal appearing 8-Cell human embryos, in comparison with published
microarrays of human fibroblasts and pluripotent stem cells.
Many genes involved in circadian rhythm and cell division were
over-expressed in the 8-Cells. The cell cycle checkpoints, RB and WEE1,
were silent on the 8-Cell arrays, whereas the recently described tumor
suppressor, UHRF2, was up-regulated > 10-fold, and the proto-oncogene,
MYC, and the core element of circadian rhythm, CLOCK, were elevated up
to > 50-fold on the 8-Cell arrays.
The canonical G1 and G2 cell cycle checkpoints are not active in
totipotent human blastomeres, perhaps replaced by UHRF2, MYC, and
intracellular circadian pathways, which may play important roles in
early human development
β-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of IL6R-Signaling in Cardiomyocytes upon Increased Workload
Excessive beta-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of beta-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium-calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the beta-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted beta-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of beta-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving beta-blockers
β-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of <i>IL6R</i>-Signaling in Cardiomyocytes upon Increased Workload
Excessive β-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of β-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium–calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the β-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted β-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of β-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving β-blockers