Islands Containing Slowly Hydrolyzable GTP Analogs Promote Microtubule Rescues

Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5′-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro

The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic

Nanotechnology intervention of the microbiome for cancer therapy

The microbiome is emerging as a key player and driver of cancer. Traditional modalities to manipulate the microbiome (for example, antibiotics, probiotics and microbiota transplants) have been shown to improve efficacy of cancer therapies in some cases, but issues such as collateral damage to the commensal microbiota and consistency of these approaches motivates efforts towards developing new technologies specifically designed for the microbiome–cancer interface. Considering the success of nanotechnology in transforming cancer diagnostics and treatment, nanotechnologies capable of manipulating interactions that occur across microscopic and molecular length scales in the microbiome and the tumour microenvironment have the potential to provide innovative strategies for cancer treatment. As such, opportunities at the intersection of nanotechnology, the microbiome and cancer are massive. In this Review, we highlight key opportunistic areas for applying nanotechnologies towards manipulating the microbiome for the treatment of cancer, give an overview of seminal work and discuss future challenges and our perspective on this emerging area

Islands containing slowly hydrolyzable GTP analogs promote microtubule rescues.

Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5'-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro

Gut commensal Enterocloster species host inoviruses that are secreted in vitro and in vivo

Abstract Background Bacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized. Results In this study, we utilized in silico, in vitro, and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. (formerly Clostridium spp.). We confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated bacterial growth dynamics, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives. Conclusion In this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria. Video Abstrac

Dynamic Light Scattering Microrheology Reveals Multiscale Viscoelasticity of Polymer Gels and Precious Biological Materials

The development of experimental techniques capable of probing the viscoelasticity of soft materials over a broad range of time scales is essential to uncovering the physics that governs their behavior. In this work, we develop a microrheology technique that requires only 12 μL of sample and is capable of resolving dynamic behavior ranging in time scales from 10^–6 to 10 s. Our approach, based on dynamic light scattering in the single-scattering limit, enables the study of polymer gels and other soft materials over a vastly larger hierarchy of time scales than macrorheology measurements. Our technique captures the viscoelastic modulus of polymer hydrogels with a broad range of stiffnesses from 10 to 10⁴ Pa. We harness these capabilities to capture hierarchical molecular relaxations in DNA and to study the rheology of precious biological materials that are impractical for macrorheology measurements, including decellularized extracellular matrices and intestinal mucus. The use of a commercially available benchtop setup that is already available to a variety of soft matter researchers renders microrheology measurements accessible to a broader range of users than existing techniques, with the potential to reveal the physics that underlies complex polymer hydrogels and biological materials

Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium

Spatial asymmetry is crucial to development. One mechanism for generating asymmetry involves the localized synthesis of a key regulatory protein that diffuses away from its source, forming a spatial gradient. Although gradients are prevalent in eukaryotes, at both the tissue and intracellular levels, it is unclear whether gradients of freely diffusible proteins can form within bacterial cells given their small size and the speed of diffusion. Here, we show that the bacterium Caulobacter crescentus generates a gradient of the active, phosphorylated form of the master regulator CtrA, which directly regulates DNA replication. Using a combination of mathematical modeling, single-cell microscopy, and genetic manipulation, we demonstrate that this gradient is produced by the polarly localized phosphorylation and dephosphorylation of CtrA. Our data indicate that cells robustly establish the asymmetric fates of daughter cells before cell division causes physical compartmentalization. More generally, our results demonstrate that uniform protein abundance may belie gradients and other sophisticated spatial patterns of protein activity in bacterial cells