ABSTRACT
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescence in situ hybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.
ABSTRACT
BACKGROUND: The incidence of type 2 diabetes is increasing disproportionately in individuals <65 years of age. It is not known whether disease characteristics in these younger patients are similar to "classic" late-onset type 2 diabetes. METHODS: In the epidemiological cohort study entitled "Retrolective Study: Self-Monitoring of Blood Glucose and Outcome in Patients with Type 2 Diabetes," a total of 3268 patients from randomly contacted primary care practices were documented during a mean follow-up period of 6.5 years. All newly diagnosed patients of these practices were included. RESULTS: At diagnosis, 64.2% of the patients were aged < or =65 years. Thereof, 57.2% were male, whereas in the age group >65 years only 35.0% were male (p < 0.001). The younger group exhibited more severe metabolic deterioration at diagnosis and in the following years than the older group. Conversely, the older group presented at diagnosis with a higher prevalence of cardiovascular risk factors. Self-monitoring of blood glucose (SMBG) was more prominent in the younger group. In both age groups, the use of SMBG was associated with a significantly lower risk (p = 0.003) of a combined end point (severe diabetic complication or all-cause mortality). CONCLUSIONS: There are considerable differences in disease characteristics between people diagnosed with type 2 diabetes during 45-65 years of age versus diagnosis at a later age. Type 2 diabetes diagnosed before the age of 65 years disproportionately affected men and exhibited a more severe disease course, but was characterized by significantly less cardiovascular risk factors in comparison to type 2 diabetes diagnosed at a later age. The use of SMBG was associated with a better clinical outcome in both age groups.
