No abstract

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed 'TRACE-seq') are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

No abstract

Cell-cell communication via gap junctions regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. Here, we developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10 s temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease.

10 Cell-cell communication via gap junctions regulates a wide range of 11 physiological processes by enabling the direct intercellular electrical and 12 chemical coupling. However, the in vivo distribution and function of gap 13 junctions remain poorly understood, partly due to the lack of non-invasive tools 14 with both cell-type specificity and high spatiotemporal resolution. Here we 15 developed PARIS (pairing actuators and receivers to optically isolate gap 16 junctions), a new fully genetically encoded tool for measuring the cell-specific 17 gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC 18 in several cultured cell lines under physiologically relevant conditions and in 19 distinct genetically defined neurons in Drosophila brain, with ~10-sec temporal 20 resolution and sub-cellular spatial resolution. These results demonstrate that 21 PARIS is a robust, highly sensitive tool for mapping functional gap junctions and 22 study their regulation in both health and disease.23 109 cell (Figure 1 -figure supplement 3). Specifically, the directly connected cell 110 had the strongest response, and the thirdly connected cell had the weakest 111 5 response (Figure 1 -figure supplement 3D). 112We then quantified the ArchT-induced pH change in the actuator cells using the 113 ratiometric pH indicator mTagBFP-pHluorinCAAX generated by fusing the pH-114 insensitive blue fluorescent protein mTagBFP (Subach et al., 2008) to the N-115 terminus of pHluorinCAAX and then calibrating the correlation between pH and 116 the ratio of GFP/BFP fluorescence (Figure 1 -figure supplement 4). Based on 117 a fit to the titration curve, we estimated that a 4-sec and 20-sec laser pulse 118 induces a transient increase of intracellular pH from 7.35 to 7.45 and 7.80 119 respectively in actuator cells (Figure 1 -figure supplement 4D-F), which allowed 120 us to repeatedly elicit a PARIS signal in specific cells as shown above. Together, 121these data provide proof-of-principle that PARIS is a robust tool for measuring 122 GJC between connected cells. 123 Electrophysiological validation of PARIS and its comparison with FRAP124 in HEK293T cells. 125 We have showed that PARIS could detect GJC in a photostimulation dependent 126 way and sensitive to CBX (Figure 2A, D1 & Figure 1). Next, we further validated 127 PARIS by patch-clamping the receiver cell in order to record the gap junction-128 mediated current induced by activating the actuator cell using a laser pulse 129 ( Figure 2B1). Applying increasingly stronger light pulses to the actuator cell 130 yielded time-locked currents in the receiver cell that were blocked by CBX 131 ( Figure 2B2,D2). In the same cells, voltage steps on the actuator cell also 132 elicited non-rectifying and CBX sensitive currents in the receiver cell ( Figure 133 2C,D3). Quantification of the group data showed that the CBX inhibition of GJC 134 was independent from the approaches used to activate the actuator cell (by 135 light or voltage) and from the signals measured in the receiver c...

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a recently developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive, and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance, and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

Huanglongbing (HLB), caused by “Candidatus liberibacter asiaticus” (CaLas), is one of the most devastating diseases in citrus but its pathogenesis remains poorly understood. Here, we reported the role of the CaLasSDE115 (CLIBASIA_05115) effector, encoded by CaLas, during pathogen-host interactions. Bioinformatics analyses showed that CaLasSDE115 was 100% conserved in all reported CaLas strains but had sequence differences compared with orthologs from other “Candidatus liberibacter.” Prediction of protein structures suggested that the crystal structure of CaLasSDE115 was very close to that of the invasion-related protein B (IalB), a virulence factor from Bartonella henselae. Alkaline phosphatase (PhoA) assay in E. coli further confirmed that CaLasSDE115 was a Sec-dependent secretory protein while subcellular localization analyses in tobacco showed that the mature protein of SDE115 (mSDE115), without its putative Sec-dependent signal peptide, was distributed in the cytoplasm and the nucleus. Expression levels of CaLasSDE115 in CaLas-infected Asian citrus psyllid (ACP) were much higher (∼45-fold) than those in CaLas-infected Wanjincheng oranges, with the expression in symptomatic leaves being significantly higher than that in asymptomatic ones. Additionally, the overexpression of mSDE115 favored CaLas proliferation during the early stages (2 months) of infection while promoting the development of symptoms. Hormone content and gene expression analysis of transgenic plants also suggested that overexpressing mSDE115 modulated the transcriptional regulation of genes involved in systemic acquired resistance (SAR) response. Overall, our data indicated that CaLasSDE115 effector contributed to the early colonization of citrus by the pathogen and worsened the occurrence of Huanglongbing symptoms, thereby providing a theoretical basis for further exploring the pathogenic mechanisms of Huanglongbing disease in citrus.

The dramatic increase in the prevalence of multi-drug resistant Gram-negative bacterial infections and the simultaneous lack of new classes of antibiotics is projected to result in approximately 10 million deaths per year by 2050. We report on efforts to target the Gram-negative ATP-binding cassette (ABC) transporter MsbA, an essential inner membrane protein that transports lipopolysaccharide from the inner leaflet to the periplasmic face of the inner membrane. We demonstrate the improvement of a high throughput screening hit into compounds with on-target single digit micromolar (μM) minimum inhibitory concentrations against wild-type uropathogenic Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. A 2.98 Å resolution X-ray crystal structure of MsbA complexed with an inhibitor revealed a novel mechanism for inhibition of an ABC transporter. The identification of a fully encapsulated membrane binding site in Gram-negative bacteria led to unique physicochemical property requirements for wild-type activity.