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Ban-Lan-Gen, the root tissues derived from several morphologically indistinguishable plant species, have been used widely in traditional Chinese medicines for numerous years. The identification of reliable markers to distinguish various source plant species is critical for the effective and safe use of products containing Ban-Lan-Gen. Here, we analyzed and characterized the complete chloroplast (cp) genome sequence of (Nees) Kuntze to identify high-resolution markers for the species determination of Southern Ban-Lan-Gen. Total DNA was extracted and subjected to next-generation sequencing. The cp genome was then assembled, and the gaps were filled using PCR amplification and Sanger sequencing. Genome annotation was conducted using CpGAVAS web server. The genome was 144,133 bp in length, presenting a typical quadripartite structure of large (LSC; 91,666 bp) and small (SSC; 17,328 bp) single-copy regions separated by a pair of inverted repeats (IRs; 17,811 bp). The genome encodes 113 unique genes, including 79 protein-coding, 30 transfer RNA, and 4 ribosomal RNA genes. A total of 20 tandem, 2 forward, and 6 palindromic repeats were detected in the genome. A phylogenetic analysis based on 65 protein-coding genes showed that was closely related to and , which belong to the same family, Acanthaceae. One interesting feature is that the IR regions apparently undergo simultaneous contraction and expansion, resulting in the presence of single copies of rps19, rpl2, rpl23, and ycf2 in the LSC region and the duplication of psbA and trnH genes in the IRs. This study provides the first complete cp genome in the genus , containing critical information for the classification of various species in the future. This study also provides the foundation for precisely determining the plant sources of Ban-Lan-Gen.

We have identified a small-molecule inhibitor of tumor necrosis factor α (TNF-α) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-α activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-α subunits.

Background Next-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods, transposed-based Nextera has a slight insertion bias. Results Here we present the discovery of a mutant transposase (Tn5-059) with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes, which leads to less sequencing and lower cost per genome. In addition, when used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50 ng input recommendation. Conclusions Enhanced DNA input tolerance of Tn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059, and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling.

Chromatin accessibility captures in vivo protein-chromosome binding status, and is considered an informative proxy for protein-DNA interactions. DNase I and Tn5 transposase assays require thousands to millions of fresh cells for comprehensive chromatin mapping. Applying Tn5 tagmentation to hundreds of cells results in sparse chromatin maps. We present a transposome hypersensitive sites sequencing assay for highly sensitive characterization of chromatin accessibility. Linear amplification of accessible DNA ends with in vitro transcription, coupled with an engineered Tn5 super-mutant, demonstrates improved sensitivity on limited input materials, and accessibility of small regions near distal enhancers, compared with ATAC-seq.

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. Next-generation sequencing technologies vary in performance, which is often measured by metrics such as sequencing speed, accuracy and read length. Here, the authors present a new sequencing by synthesis method that monitors polymerase binding to DNA, and suggest that this method has the potential to generate longer and faster reads.

We have identified a small-molecule inhibitor of tumor necrosis factor [alpha] (TNF-[alpha]) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-[alpha] activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-[alpha] subunits.

Non-small cell lung cancer (NSCLC) frequently develops resistance to tyrosine kinase inhibitors (TKIs), limiting the long-term success of targeted therapies. A deeper understanding of resistance mechanisms at the molecular and cellular levels may enable the development of more effective treatment strategies. Here, we applied the Teton detection assay on the AVITI24 platform to measure concurrently RNA, protein, and cellular morphology in NSCLC cell lines treated with the TKIs gefitinib and osimertinib. This single-cell, multiomic analysis revealed distinct expression and morphological profiles between drug-sensitive and resistant cells, including differences in MAPK-related pathway activity. Stratifying responses at the single-cell level uncovered subtle responses not detectable in bulk measurements. We identified CDK4/6 activity as a route of cell survival under TKI treatment and demonstrated that co-treatment with the CDK4/6 inhibitor palbociclib enhanced TKI efficacy. The ability to measure multiomics and cellular morphology in the same cells opens new avenues for future studies aimed at improving personalized treatment strategies in NSCLC and overcoming the obstacles posed by drug resistance.

We present a multiomics platform comprising Teton, a detection assay system, and AVITI24, a dual-flowcell instrument that performs both cellular imaging and sequencing readout. Teton integrates a compartmentalized flowcell for cell culture with methods to measure morphology, RNA, and protein at subcellular resolution. The platform quantifies morphological features through cell painting of 6 cellular components, RNA expression of up to 350 transcripts via sequencing of oligonucleotides hybridized to mRNA, and protein expression of up to 200 targets using antibody-linked oligonucleotide sequencing. The flow cell accommodates >1 million cells in a 10 cm sqaured open-well format or can be subdivided into 12 or 48 wells to support experiments with multiple conditions or time points. We describe and validate the detection methods of the platform and showcase its capabilities by co-culturing three cancer cell lines and elucidating the cellular pathways triggered by various drug treatments as a function of time. Using multiple time points enables us to capture the dynamics of cellular processes including receptor activation and signaling cascades. The results demonstrate how different cancer cells evade TNFalpha-induced apoptosis by activating compensatory signaling programs that maintain survival despite pro-apoptotic cues. Our model system replicates previously published results and highlights the versatility of the platform in enabling rapid, high-throughput analysis of complex cellular responses in varied biological contexts.

The lactose permease of Escherichia coli is a paradigm for understanding the structure and mechanism of many membrane transport proteins. The overall focus of this dissertation is to obtain both static and dynamic structure information on this protein. Cys-scanning mutagenesis has been employed systematically to examine residues in putative helix XII and the flanking periplasmic loop. None of the residues plays a central role in the transport mechanism. However, the results demonstrate that the cytoplasmic end of the putative helix is important for protein stabilization. Site-directed spin labeling was employed to further characterize structure features of putative helix XII. The results confirm that transmembrane domain XII is indeed in$\\alpha$ -helical conformation with one face of the helix in contact with the remainder of the protein and the other face in contact with the bilayer. Engineering divalent metal-binding sites was used as a novel approach to obtain helix packing information. By this means, bis-His residues are introduced at positions thought to be close to each other and electron paramagnetic resonance spectroscopy is used to measure divalent metal binding.$\\rm Mn\\sp{2+}$binding by the double mutant$\\rm Asp237\\to His$(helix VII)/ $\\rm Lys358\\to His$(helix XI) provides the first direct evidence that the two functionally interacting charge residues are in close physical proximity. The interactions between the four irreplaceable residues in the permease, Glu269(helix VIII) with His322(helix X) and Arg302(helix IV) with Glu325(helix X), have been confirmed and extended by engineering bis- or tris-His residues between$\\rm Glu269\\to His$and His322;$\\rm Arg302\\to His$and His322;$\\rm Glu325\\to His,\\ Arg302\\to His$and His322, and most recently,$\\rm Glu269\\to His$and$\\rm Arg302\\to His.$The results are in agreement with a mechanism model in which all four essential residues are in close proximity and undergo changes in interaction with each other during turnover. Site-directed fluorescence spectroscopy demonstrates that the interaction between Glu269 and His322 plays important role in stabilizing the substrate binding interface between helices V and VIII. Finally, it is demonstrated that substrate stabilizes the protein against heat denaturation and that the permease can be refolded in vitro after chemical denaturation.