RESUMO
The first step in SARS-CoV-2 genomic surveillance is testing to identify infected people. However, global testing rates are falling as we emerge from the acute health emergency and remain low in many low- and middle-income countries (LMICs) (mean = 27 tests/100,000 people/day). We simulated COVID-19 epidemics in a prototypical LMIC to investigate how testing rates, sampling strategies, and sequencing proportions jointly impact surveillance outcomes and showed that low testing rates and spatiotemporal biases delay time-to-detection of new variants by weeks-to-months and can lead to unreliable estimates of variant prevalence even when the proportion of samples sequenced is increased. Accordingly, investments in wider access to diagnostics to support testing rates of [~]100 tests/100,000 people/day could enable more timely detection of new variants and reliable estimates of variant prevalence. The performance of global SARS-CoV-2 genomic surveillance programs is fundamentally limited by access to diagnostic testing.
RESUMO
The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.
Assuntos
Reparo do DNA , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Reagentes de Ligações Cruzadas/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Endonucleases/química , Endonucleases/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Fotoquímica , Ligação Proteica , Conformação Proteica , Sequências de Repetição em Tandem/genética , Fatores de TempoRESUMO
Synthetic glucocorticoids belong to the most frequently administered drugs in livestock production. These synthetic hormones are employed for therapeutic purposes against inflammatory reactions, disorders of the musculoskeletal system, bovine ketosis and many other diseases of farm animals. A widespread illegal use of synthetic glucocorticoids to improve feed intake and weight gain has also been observed. To enforce the residue limits imposed on glucocorticoid drugs and preclude their illicit administration as growth promoters, it is necessary to establish high throughput analytical methods that can be applied to the screening of animal tissues. Here, we developed a dual luciferase reporter assay that detects residues or contaminants with glucocorticoid activity. This screening assay is performed by transfection of human cell lines with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for assay variabilities and unspecific matrix effects. The limit of detection (1.25 microg for dexamethasone in liver) depends on the biological potency of each synthetic glucocorticoid but, with all drugs tested, the maximal response reaches a 20 to 30 fold induction of luciferase activity. In combination with an appropriate sample clean-up method (recovery of 82%), this luciferase assay has been applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual luciferase reporter assay provides a new screening tool to detect unwanted glucocorticoid activities in animal tissues or other crude biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites.
