Biology subjects

Thomas, D.

Publications and source records attributed to Thomas, D..

60 records · Page 4Linked to original sources

bioRxiv2017

Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

Fidaxomicin is an antibacterial drug in clinical use in treatment of Clostridium difficile diarrhea1-2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1-4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1-2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6-8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9 and inhibits transcription initiation at an earlier step than Rif8-11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12-13 structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5 [A] resolution. The structure shows that Lpm binds at the base of the RNAP \"clamp,\" interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17{degrees} relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14 experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor {sigma} regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

molecular biology↗

bioRxiv2017

Rolling a mycobiome down a hill: endophytes in the Taiwanese Cloud Forest

Fungal endophytes of plants are ubiquitous and important to host plant health. Despite their ecological importance, landscape-level patterns of microbial communities in plant hosts are not well-characterized. Fungal wood-inhabiting and foliar endophyte communities from multiple tree hosts were sampled at multiple spatial scales across a 25 ha subtropical research plot in northern Taiwan, using culture-free, community DNA amplicon sequencing methods. Fungal endophyte communities were distinct between leaves and wood, but the mycobiomes were highly variable across and within tree species. Of the variance that could be explained, host tree species was the most important driver of mycobiome community-composition. Within a single tree species, \"core\" mycobiomes were characterized using cooccurrence analysis. These core groups of endophytes in leaves and wood show divergent spatial patterns. For wood endophytes, a more consistent, \"minimal\" core mycobiome coexisted with the host across the extent of the study. For leaf endophytes, the core fungi resembled a more dynamic, \"gradient\" model of the core microbiome, changing across the topography and distance of the study.

microbiology↗

bioRxiv2017

Non-invasive measurement of hepatic venous oxygen saturation (ShvO2) with quantitative susceptibility mapping in normal mouse liver and livers bearing colorectal metastases

PurposeThe purpose of this prospective study was to investigate the potential of QSM to non-invasively measure hepatic venous oxygen saturation (ShvO2).\n\nMaterials & MethodsAll animal studies were performed in accordance with the UK Home Office Animals Science Procedures Act (1986) and UK National Cancer Research Institute (NCRI) guidelines. QSM data was acquired from a cohort of mice (n=10) under both normoxic (medical air, 21% O2/ balance N), and hyperoxic conditions (100% O2). Susceptibility measurements were taken from large branches of the portal and hepatic vein under each condition and were used to calculate venous oxygen saturation in each vessel. Blood was extracted from the IVC of three mice under norm- and hyperoxic conditions, and oxygen saturation was measured using a blood gas analyser to act as a gold standard. QSM data was also acquired from a cohort of mice bearing colorectal liver metastases (CRLM). SvO2 was calculated from susceptibility measurements made in the portal and hepatic veins, and compared to the healthy animals.\n\nResultsSvO2 calculated from QSM measurements showed a significant increase of 14.93% in the portal vein (p < 0.05), and an increase of 21.39% in the hepatic vein (p < 0.01). Calculated results showed excellent agreement with those from the blood gas analyser (26.14% increase). ShvO2 was significantly lower in the disease cohort (30.18 {+/-} 11.6%), than the healthy animals (52.67 {+/-} 17.8%) (p < 0.05), but differences in the portal vein were not significant.\n\nConclusionQSM is a feasible tool for non-invasively measuring hepatic venous oxygen saturation and can detect differences in oxygen consumption in livers bearing colorectal metastases.

biophysics↗

bioRxiv2017

Harnessing the adaptive potential of mechanoresponsive proteins to overwhelm pancreatic cancer dissemination and invasion

Metastatic disease is often characterized by altered cellular contractility and deformability, lending cells and groups of cells the flexibility to navigate through different microenvironments. This ability to change cell shape is driven in large part by the structural elements of the mechanobiome, which includes cytoskeletal proteins that sense and respond to mechanical stimuli. Here, we demonstrate that key mechanoresponsive proteins (those which accumulate in response to mechanical stress), specifically nonmuscle myosin IIA and IIC, -actinin 4, and filamin B, are highly upregulated in pancreatic ductal adenocarcinoma cancer (PDAC) and in patient-derived pancreatic cancer cell lines. Their less responsive sister paralogs (myosin IIB, -actinin 1, and filamin A) show a smaller dynamic range or disappear with PDAC progression. We demonstrate that these mechanoresponsive proteins directly impact cell mechanics using knock-down and overexpression cell lines. We further quantify the nonmuscle myosin II family members in patient-derived cell lines and identify a role for myosin IIC in the formation of transverse actin arcs in single cells and cortical actin belts in tissue spheroids. We harness the upregulation of myosin IIC and its impact of cytoskeletal architecture through the use of the mechanical modulator 4-hydroxyacetophenone (4-HAP), which increases myosin IIC assembly and stiffens cells. Here, 4-HAP decreases dissemination, induces cortical actin belts, and slows retrograde actin flow in spheroids. Finally, mice having undergone hemi-splenectomies with PDAC cells and then treated with 4-HAP have a reduction in liver metastases. Thus, increasing the activity of these mechanoresponsive proteins (in this case, by increasing myosin IIC assembly) to overwhelm the ability of cells to polarize and invade may be an effective strategy to improve the five-year survival rate of pancreatic cancer patients, currently hovering around 6%.

cancer biology↗

bioRxiv2017

Understanding and mitigating some limitations of Illumina (C) MiSeq for environmental sequencing of fungi.

ITS-amplicon metabarcode studies using the illumina MiSeq sequencing platform are the current standard tool for fungal ecology studies. Here we report on some of the particular challenges experienced while creating and using a ribosomal RNA gene (rDNA) amplicon library for an ecological study. Two significant complications were encountered. First, artificial differences in read abundances among OTUs were observed, apparently resulting from bias at two stages: PCR amplification of genomic DNA with ITS-region Illumina-sequence-adapted-primers, and during Illumina sequencing. These differential read abundances were only partially corrected by a common variance-stabilization method. Second, tag-switching (or the shifting of amplicons to incorrect sample indices) occurred at high levels in positive mock-community controls. An example of a bioinformatic method to estimate the rate of tag switching is shown, some recommendations on the use of positive controls and primer choice are given, and one approach to reducing potential false positives resulting from these technological biases is presented.

bioinformatics↗

bioRxiv2017

Fine-mapping of genetic loci driving spontaneous clearance of hepatitis C virus infection

Approximately three quarters of acute HCV infections evolve to a chronic state, while one quarter are spontaneously cleared. Genetic predispositions strongly contribute to the development of chronicity. We have conducted a genome-wide association study to identify genomic variants underlying HCV spontaneous clearance using Immunochip in European and African ancestries. We confirmed two previously reported significant associations, in the IL28B/IFNL41,2 and MHC regions, with spontaneous clearance in the European population. We further fine-mapped the MHC association to a region of about 50 kilo base pairs, down from 1 mega base pairs in the previous study. Additional analyses suggested that the association in the MHC locus might be significantly stronger for virus subtype 1a than 1b, suggesting that viral subtype may have influenced the genetic mechanism underlying the clearance of HCV.

immunology↗