Protocol for isolating small cytosolic dsDNA from cultured murine cells.

We recently identified a class of small cytosolic double-stranded DNA (scDNA) approximately 20-40 bp in size in human and mouse cells. Here, we present a protocol for scDNA isolation from cultured murine cells. We describe steps for cytosolic compartment separation, DNA isolation in the cytosolic fraction using phenol-chloroform extraction, and ethanol precipitation. We then detail procedures for denaturing purified cytosolic DNA through urea polyacrylamide gel electrophoresis and obtaining scDNA in the cytosolic DNA fraction via gel purification. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Neutralization of EG.5, EG.5.1, BA.2.86, and JN.1 by antisera from dimeric receptor-binding domain subunit vaccines and 41 human monoclonal antibodies.

BACKGROUND: The recently circulating Omicron variants BA.2.86 and JN.1 were identified with more than 30 amino acid changes on the spike protein compared to BA.2 or XBB.1.5. This study aimed to comprehensively assess the immune escape potential of BA.2.86, JN.1, EG.5, and EG.5.1. METHODS: We collected human and murine sera to evaluate serological neutralization activities. The participants received three doses of coronavirus disease 2019 (COVID-19) vaccines or a booster dose of the ZF2022-A vaccine (Delta-BA.5 receptor-binding domain [RBD]-heterodimer immunogen) or experienced a breakthrough infection (BTI). The ZF2202-A vaccine is under clinical trial study (ClinicalTrials.gov: NCT05850507). BALB/c mice were vaccinated with a panel of severe acute respiratory syndrome coronavirus 2 RBD-dimer proteins. The antibody evasion properties of these variants were analyzed with 41 representative human monoclonal antibodies targeting the eight RBD epitopes. FINDINGS: We found that BA.2.86 had less neutralization evasion than EG.5 and EG.5.1 in humans. The ZF2202-A booster induced significantly higher neutralizing titers than BTI. Furthermore, BA.2.86 and JN.1 exhibited stronger antibody evasion than EG.5 and EG.5.1 on RBD-4 and RBD-5 epitopes. Compared to BA.2.86, JN.1 further lost the ability to bind to several RBD-1 monoclonal antibodies and displayed further immune escape. CONCLUSIONS: Our data showed that the currently dominating sub-variant, JN.1, showed increased immune evasion compared to BA.2.86 and EG.5.1, which is highly concerning. This study provides a timely risk assessment of the interested sub-variants and the basis for updating COVID-19 vaccines. FUNDING: This work was funded by the National Key R&D Program of China, the National Natural Science Foundation of China, the Beijing Life Science Academy, the Bill & Melinda Gates Foundation, and the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (CPSF).

Room-Temperature Growth of Square-Millimeter Single-Crystalline Two-Dimensional Metal Halides on Silicon.

Silicon is the cornerstone of electronics and photonics. In this context, almost all integrated devices derived from two-dimensional (2D) materials stay rooted in silicon technology. However, as the growth substrate, silicon has long been thought to be a hindrance for growing 2D materials through bottom-up methods that require high growth temperatures, and thus, indirect routes are usually considered instead. Although promising growth of large-area 2D materials on silicon has been demonstrated, the direct growth of single-crystalline materials using low-thermal-budget synthesis methods remains challenging. Here, we report the room-temperature growth of millimeter-scale single-crystal 2D metal halides on silicon substrates with a hydroxyl-terminated surface. Theoretical calculations reveal that the activation energy for surface diffusion can be reduced by an order of magnitude by terminating the surface with hydroxyl groups, from which on-silicon growth is greatly facilitated at room temperature and enables a 4-order-of-magnitude increase in area. The high quality and uniformity of the resulting single crystals are further evidenced. The optoelectronic devices employing the as-grown materials show an ultralow dark current of 10-13 A and a high detectivity of 1013 Jones, thereby corroborating a weak-light detection ability. These results would point to a rich space of surface modulation that can be used to surmount current limitations and demonstrate a promising strategy for growing 2D materials directly on silicon at room temperature to produce large single crystals.

SARS-CoV-2 N protein-induced Dicer, XPO5, SRSF3, and hnRNPA3 downregulation causes pneumonia.

Though RNAi and RNA-splicing machineries are involved in regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, their precise roles in coronavirus disease 2019 (COVID-19) pathogenesis remain unclear. Herein, we show that decreased RNAi component (Dicer and XPO5) and splicing factor (SRSF3 and hnRNPA3) expression correlate with increased COVID-19 severity. SARS-CoV-2 N protein induces the autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, inhibiting miRNA biogenesis and RNA splicing and triggering DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 knockdown increases, while their overexpression decreases, N protein-induced pneumonia's severity. Older mice show lower expression of Dicer, XPO5, SRSF3, and hnRNPA3 in their lung tissues and exhibit more severe N protein-induced pneumonia than younger mice. PJ34, a poly(ADP-ribose) polymerase inhibitor, or anastrozole, an aromatase inhibitor, ameliorates N protein- or SARS-CoV-2-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.

Mutual communication between radiosensitive and radioresistant esophageal cancer cells modulates their radiosensitivity.

Radiotherapy is an important treatment modality for patients with esophageal cancer; however, the response to radiation varies among different tumor subpopulations due to tumor heterogeneity. Cancer cells that survive radiotherapy (i.e., radioresistant) may proliferate, ultimately resulting in cancer relapse. However, the interaction between radiosensitive and radioresistant cancer cells remains to be elucidated. In this study, we found that the mutual communication between radiosensitive and radioresistant esophageal cancer cells modulated their radiosensitivity. Radiosensitive cells secreted more exosomal let-7a and less interleukin-6 (IL-6) than radioresistant cells. Exosomal let-7a secreted by radiosensitive cells increased the radiosensitivity of radioresistant cells, whereas IL-6 secreted by radioresistant cells decreased the radiosensitivity of radiosensitive cells. Although the serum levels of let-7a and IL-6 before radiotherapy did not vary significantly between patients with radioresistant and radiosensitive diseases, radiotherapy induced a more pronounced decrease in serum let-7a levels and a greater increase in serum IL-6 levels in patients with radioresistant cancer compared to those with radiosensitive cancer. The percentage decrease in serum let-7a and the percentage increase in serum IL-6 levels at the early stage of radiotherapy were inversely associated with tumor regression after radiotherapy. Our findings suggest that early changes in serum let-7a and IL-6 levels may be used as a biomarker to predict the response to radiotherapy in patients with esophageal cancer and provide new insights into subsequent treatments.