Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation

Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo. Keywords: cancer genetics; cancer models; cancer therapeutic resistance; gene regulation; CRISPRNational Cancer Institute (U.S.) (Grant U54-CA112967-06)National Cancer Institute (U.S.) (Grant P30-CA14051

BCL-2 family genetic profiling reveals microenvironment-specific determinants of chemotherapeutic response

The Bcl-2 family encompasses a diverse set of apoptotic regulators that are dynamically activated in response to various cell-intrinsic and -extrinsic stimuli. An extensive variety of cell culture experiments have identified effects of growth factors, cytokines, and drugs on Bcl-2 family functions, but in vivo studies have tended to focus on the role of one or two particular members in development and organ homeostasis. Thus, the ability of physiologically relevant contexts to modulate canonical dependencies that are likely to be more complex has yet to be investigated systematically. In this study, we report findings derived from a pool-based shRNA assay that systematically and comprehensively interrogated the functional dependence of leukemia and lymphoma cells upon various Bcl-2 family members across many diverse in vitro and in vivo settings. This approach permitted us to report the first in vivo loss of function screen for modifiers of the response to a front-line chemotherapeutic agent. Notably, our results reveal an unexpected role for the extrinsic death pathway as a tissue-specific modifier of therapeutic response. In particular, our findings show that particular tissue sites of tumor dissemination play critical roles in demarcating the nature and extent of cancer cell vulnerabilities and mechanisms of chemoresistance. Cancer Res; 71(17); 5850–8. ©2011 AACR.National Institutes of Health (U.S.) (NIH RO1 CA128803)National Cancer Institute (U.S.) (Integrated Cancer Biology Program grant NCI 1-U54-CA112967)David H. Koch Institute for Integrative Cancer Research at MIT (Ludwig Fellowship)Massachusetts Institute of Technology. Dept. of Biology (training grant

Decreased microbial co-occurrence network stability and SCFA receptor level correlates with obesity in African-origin women.

We compared the gut microbial populations in 100 women, from rural Ghana and urban US [50% lean (BMI < 25 kg/m2) and 50% obese (BMI ≥ 30 kg/m2)] to examine the ecological co-occurrence network topology of the gut microbiota as well as the relationship of short chain fatty acids (SCFAs) with obesity. Ghanaians consumed significantly more dietary fiber, had greater microbial alpha-diversity, different beta-diversity, and had a greater concentration of total fecal SCFAs (p-value < 0.002). Lean Ghanaians had significantly greater network density, connectivity and stability than either obese Ghanaians, or lean and obese US participants (false discovery rate (FDR) corrected p-value ≤ 0.01). Bacteroides uniformis was significantly more abundant in lean women, irrespective of country (FDR corrected p < 0.001), while lean Ghanaians had a significantly greater proportion of Ruminococcus callidus, Prevotella copri, and Escherichia coli, and smaller proportions of Lachnospiraceae, Bacteroides and Parabacteroides. Lean Ghanaians had a significantly greater abundance of predicted microbial genes that catalyzed the production of butyric acid via the fermentation of pyruvate or branched amino-acids, while obese Ghanaians and US women (irrespective of BMI) had a significantly greater abundance of predicted microbial genes that encoded for enzymes associated with the fermentation of amino-acids such as alanine, aspartate, lysine and glutamate. Similar to lean Ghanaian women, mice humanized with stool from the lean Ghanaian participant had a significantly lower abundance of family Lachnospiraceae and genus Bacteroides and Parabacteroides, and were resistant to obesity following 6-weeks of high fat feeding (p-value < 0.01). Obesity-resistant mice also showed increased intestinal transcriptional expression of the free fatty acid (Ffa) receptor Ffa2, in spite of similar fecal SCFAs concentrations. We demonstrate that the association between obesity resistance and increased predicted ecological connectivity and stability of the lean Ghanaian microbiota, as well as increased local SCFA receptor level, provides evidence of the importance of robust gut ecologic network in obesity

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.National Human Genome Research Institute (U.S.) (Grant P50HG006193

Health-related quality of life after treatment for bladder cancer in England

Background Little is known about quality of life after bladder cancer treatment. This common cancer is managed using treatments that can affect urinary, sexual and bowel function. Methods To understand quality of life and inform future care, the Department of Health (England) surveyed adults surviving bladder cancer 1–5 years after diagnosis. Questions related to disease status, co-existing conditions, generic health (EQ-5D), cancer-generic (Social Difficulties Inventory) and cancer-specific outcomes (Functional Assessment of Cancer Therapy—Bladder). Results In total, 673 (54%) patients responded; including 500 (74%) men and 539 (80%) with co-existing conditions. Most respondents received endoscopic treatment (60%), while 92 (14%) and 99 (15%) received radical cystectomy or radiotherapy, respectively. Questionnaire completion rates varied (51–97%). Treatment groups reported ≥1 problem using EQ-5D generic domains (59–74%). Usual activities was the most common concern. Urinary frequency was common after endoscopy (34–37%) and radiotherapy (44–50%). Certain populations were more likely to report generic, cancer-generic and cancer-specific problems; notably those with co-existing long-term conditions and those treated with radiotherapy. Conclusion The study demonstrates the importance of assessing patient-reported outcomes in this population. There is a need for larger, more in-depth studies to fully understand the challenges patients with bladder cancer face

Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information. © 2013 Czapla et al

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth

Structure and lipid binding properties of the kindlin-3 pleckstrin homology domain

Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at 2.23Å resolution and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics (MD) simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP) binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1-β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared to PtdIns(4,5)P2 Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as analyte indicate affinities of 300 μM for PtdIns(3,4,5)P3 and 1mM for PtdIns(4,5)P2 In contrast, SPR studies with immobilised PH domain and lipid nanodiscs as analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (~5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1-β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance lipid mobility and clustering for the biophysical assessment of protein-membrane interactions

Additive Manufacturing of Transparent Fused Quartz

This paper investigates a filament-fed process for additive manufacturing (AM) of fused quartz. Glasses such as fused quartz have significant scientific and engineering applications, which include optics, communications, electronics, and hermetic seals. AM has several attractive benefits such as increased design freedom, faster prototyping, and lower processing costs for small production volumes. However, current research into glass AM has focused primarily on nonoptical applications. Fused quartz is studied here because of its desirability for use in high-quality optics due to its high transmissivity and thermal stability. Fused quartz filaments are fed into a CO2 laser-generated molten region, smoothly depositing material onto the workpiece. Spectroscopy and pyrometry are used to measure the thermal radiation incandescently emitted from the molten region. The effects of the laser power and scan speed are determined by measuring the morphology of single tracks. Thin walls are printed to study the effects of layer-to-layer height. This information is used to deposit solid pieces including a cylindrical-convex shape capable of focusing visible light. The transmittance and index homogeneity of the printed fused quartz are measured. These results show that the filament-fed process has the potential to print transmissive optics

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth