Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes.

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub.

Asymmetric cell division (ACD) produces morphologically and behaviorally distinct cells and is the primary way to generate cell diversity. In the model bacterium Caulobacter crescentus, the polarization of distinct scaffold-signaling hubs at the swarmer and stalked cell poles constitutes the basis of ACD. However, mechanisms involved in the formation of these hubs remain elusive. Here, we show that a swarmer-cell-pole scaffold, PodJ, forms biomolecular condensates both in vitro and in living cells via phase separation. The coiled-coil 4-6 and the intrinsically disordered regions are the primary domains that contribute to biomolecular condensate generation and signaling protein recruitment in PodJ. Moreover, a negative regulation of PodJ phase separation by the stalked-cell-pole scaffold protein SpmX is revealed. SpmX impedes PodJ cell-pole accumulation and affects its recruitment ability. Together, by modulating the assembly and dynamics of scaffold-signaling hubs, phase separation may serve as a general biophysical mechanism that underlies the regulation of ACD in bacteria and other organisms.