First-in-class Microbial Ecosystem Therapeutic 4 (MET4) in combination with immune checkpoint inhibitors in patients with advanced solid tumors (MET4-IO trial)

Background: The intestinal microbiome has been associated with response to immune checkpoint inhibitors (ICIs) in humans and causally implicated in ICI responsiveness in animal models. Two recent human trials demonstrated that fecal microbiota transplant (FMT) from ICI responders can rescue ICI responses in refractory melanoma, but FMT has specific limitations to scaled use.Patients and methods: We conducted an early-phase clinical trial of a cultivated, orally delivered 30-species microbial consortium (Microbial Ecosystem Therapeutic 4, MET4) designed for co-administration with ICIs as an alternative to FMT and assessed safety, tolerability and ecological responses in patients with advanced solid tumors.Results: The trial achieved its primary safety and tolerability outcomes. There were no statistically significant differences in the primary ecological outcomes; however, differences in MET4 species relative abundance were evident after randomization that varied by patient and species. Increases in the relative abundance of several MET4 taxa, including Enterococcus and Bifidobacterium, taxa previously associated with ICI responsiveness, were observed and MET4 engraftment was associated with decreases in plasma and stool primary bile acids.Conclusions: This trial is the first report of the use of a microbial consortium as an alternative to FMT in advanced cancer patients receiving ICI and the results justify the further development of microbial consortia as a therapeutic co-intervention for ICI treatment in cancer

Sheep Updates 2005 - Part 3

This session covers seven papers from different authors: CUSTOMER 1. Benefits VIAscanR to producers and WAMMCO, Rob Davidson, Supply Development Manager, David Pethick, School of Veterinary and Biomedical Studies, Murdock University. 2. Healthy fats in lamb: how WA lambs compare with others, C. F. Engelke Animal Biology, University of Western Australia, bCSIRO Livestock Industries, Western Australia B.D. Siebert, Department of Animal Science, University of Adelaide, South Australia, K. Gregg, Centre for High-Throughput Agricultural Genetic Analysis, Murdoch University, Western Australia. A-D.G. Wright CSIRO Livestock Industries, Western Australia, P.E Vercoe Animal Biology, University of Western Australia 3. Shelf life of fresh lamb meat: lamb age & electrical stimulation, Dr Robin Jacob, Department of Agriculture, Western Australia 4. Pastures from space - An evaluation of adoption of by Australian woolgrowers, Russell Barnett, Australian Venture Consultants, Joanne Sneddon, University of Western Australia 5. Your clients can learn from ASHEEP\u27s example, Sandra Brown Department of Agriculture Western Australia 6. Lifetime Wool - Farmers attitudes affect their adoption of recommended ewe management, G. Rose Department of Agriculture Western Australia, C. Kabore, Kazresearch, Lower Templestowe Vic, J. Dart, Clear Horizons, Hastings Vic 7. Sustainable certification of Australian Merino, what will customers be looking for? Stuart Adams, i-merino / iZWool International Pty Lt

Type I-F CRISPR-Cas resistance against virulent phages results in abortive infection and provides population-level immunity

Funder: Veni grant, Netherlands Organization for Scientific Research (NWO) [016.Veni.171.047 to RHJS] Health Sciences Career Development Award from the University of Otago, NZAbstract: Type I CRISPR-Cas systems are abundant and widespread adaptive immune systems in bacteria and can greatly enhance bacterial survival in the face of phage infection. Upon phage infection, some CRISPR-Cas immune responses result in bacterial dormancy or slowed growth, which suggests the outcomes for infected cells may vary between systems. Here we demonstrate that type I CRISPR immunity of Pectobacterium atrosepticum leads to suppression of two unrelated virulent phages, ɸTE and ɸM1. Immunity results in an abortive infection response, where infected cells do not survive, but viral propagation is severely decreased, resulting in population protection due to the reduced phage epidemic. Our findings challenge the view of CRISPR-Cas as a system that protects the individual cell and supports growing evidence of abortive infection by some types of CRISPR-Cas systems

Considerations for best practices in studies of fiber or other dietary components and the intestinal microbiome

Considerations for best practices in studies of fiber or other dietary components and the intestinal microbiome. Am J Physiol Endocrinol Metab 315: E1087–E1097, 2018. First published August 21, 2018; doi:10.1152/ajpendo.00058.2018.—A 2-day workshop organized by the National Institutes of Health and U.S. Department of Agriculture included 16 presentations focused on the role of diet in alterations of the gastrointestinal microbiome, primarily that of the colon. Although thousands of research projects have been funded by U.S. federal agencies to study the intestinal microbiome of humans and a variety of animal models, only a minority addresses dietary effects, and a small subset is described in sufficient detail to allow reproduction of a study. Whereas there are standards being developed for many aspects of microbiome studies, such as sample collection, nucleic acid extraction, data handling, etc., none has been proposed for the dietary component; thus this workshop focused on the latter specific point. It is important to foster rigor in design and reproducibility of published studies to maintain high quality and enable designs that can be compared in systematic reviews. Speakers addressed the influence of the structure of the fermentable carbohydrate on the microbiota and the variables to consider in design of studies using animals, in vitro models, and human subjects. For all types of studies, strengths and weaknesses of various designs were highlighted, and for human studies, comparisons between controlled feeding and observational designs were discussed. Because of the lack of published, best-diet formulations for specific research questions, the main recommendation is to describe dietary ingredients and treatments in as much detail as possible to allow reproduction by other scientists

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by “an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. Results: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Conclusions: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems

Enterohemorrhagic E. coli Requires N-WASP for Efficient Type III Translocation but Not for EspFU-Mediated Actin Pedestal Formation

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspFU (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspFU is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspFU into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspFU, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspFU were the only bacterial effectors required for pedestal formation, and the EspFU sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspFU, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008435)National Centers for Systems Biology (U.S.) (Grant 1P50GM098792)United States. Defense Threat Reduction Agency (HDTRA1-14-1-0007)Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF13D0001)National Institute of General Medical Sciences (U.S.) (Interdepartmental Biotechnology Training Program 5T32 GM008334)Fonds de la recherche en sante du Quebec (Master's Training Award

Sheep Updates 2007 - part 4

This session covers eight papers from different authors: GRAZING 1. The impact of high dietary salt and its implications for the management of livestock grazing saline land, Dean Thomas, Dominique Blache, Dean Revell, Hayley Norman, Phil Vercoe, Zoey Durmic, Serina Digby, Di Mayberry, Megan Chadwick, Martin Sillence and David Masters, CRC for Plant-based Management of Dryland Salinity, Faculty of Natural & Agricultural Sciences, The University of Western Australia, WA. 2. Sustainable Grazing on Saline Lands - outcomes from the WA1 research project, H.C. Norman1,2, D.G. Masters1,2, R. Silberstein1,2, F. Byrne2,3, P.G.H. Nichols2,4, J. Young3, L. Atkins1,2, M.G. Wilmot1,2, A.J. Rintoul1,2, T. Lambert1,2, D.R. McClements2,4, P. Raper4, P. Ward1,2, C. Walton5 and T. York6 1CSIRO Centre for Environment and Life Sciences, Wembley, WA 2CRC for Plant-based Management of Dryland Salinity. 3School of Agricultural and Resource Economics, University of Western Australia. 4Department of Agriculture and Food WA. 5Condering Hills, Yealering. 6Anameka Farms, Tammin. MEAT QUALITY 3. Development of intramuscular fat in prime lambs, young sheep and beef cattle, David Pethick1, David Hopkins2 and Malcolm McPhee3,1School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA, 2NSW Department of Primary Industries, Cowra, NSW,3NSW Dept. of Primary Industries, University of New England, Armidale, NSW, 4. Importance of drinking water temperature for managing heat stress in sheep, Savage DB, Nolan JV, Godwin IR, Aoetpah A, Nguyen T, Baillie N and Lawler C University of New England, Armidale, NSW, Australia EWE MANAGEMENT TOOLS 5. E - sheep Management of Pregnant Merino Ewes and their Finishing Lambs, Ken GeentyA, John SmithA, Darryl SmithB, Tim DyallA and Grant UphillA A Sheep CRC and CSIRO Livestock Industries, Chiswick, NSW B Turretfield Research Station, SARDI, Roseworthy, SA 6. Is it important to manage ewes to CS targets? John Young, Farming Systems Analysis Service, Kojonup, WA MULESING 7. Mulesing accreditation - Vital for Wool\u27s Future, Dr Michael Paton, Department of Agriculture and Food WA, 8. Mulesing Alternatives, Jules Dorrian, Affiliation Project Manager Blowfly Control Australian Wool Inovatio