Impacts of Forage Cropping Decisions on Feed-Flows in Cool- Temperate Grazing Systems

Intensive cool-temperate grasslands often rely on adding forage crops to help supply feed when pasture growth is limited by cool temperatures and low sunlight. We tested the impacts of using single- or multi-graze crops to alter feed supply in either summer/autumn or winter on productivity in red deer farming systems in a cool-temperate environment. The choice of single-graze or multi-graze crops to provide forage in deer grazing systems had an impact on the feed flows and the amount of pasture available, with multi-graze crops supplying more feed during the dry summer months while single-graze crops supplying more feed during the cool winter months. The limited growth rates of young red deer, and the late calving of hinds meant that pasture covers could be low during the late winter and early spring without compromising animal production. Overall, the use of a multi-graze crop, especially when used early in autumn to promote weaner liveweight gain, increased overall productivity by 5% and increased feed conversion efficiency by 2.5% in the venison production system

Purification, Mechanism, and Biochemical Interactions of the Essential Mycolic Acid Transporters MmpL3 from Mycobacterium tuberculosis, and CmpL1 from Corynebacterium glutamicum.

The Corynebacterium – Mycobacterium – Nocardia family (CMN family) are a subset group of actinomycetes which are characterized as acid-fast, Gram-positive species. Many members of the CMN family are causative agents of human disease with M. tuberculosis, M. leprae, C. diptheria, and N. asteroides historically being exceptionally hard to treat pathogens. One of the reasons the CMN family are resistant to treatment is also the defining trait of the family, which is the presence of an outer membrane with a unique composition of peptidoglycan crosslinked to an arabinogalactan matrix which is further crosslinked to mycolic acids forming an effective outer barrier. This peptidoglycan – arabinogalactan – mycolic acid barrier forms the inner leaflet of the outer membrane, with loosely associated mycolic acids esterified to disaccharide trehalose sugars forming the outer leaflet of the outer membrane. Both of these structural features together form the ‘myco-membrane’ which is even further complicated by the presence of an outer capsular layer comprised mainly of extended sugar polymers and proteins which are thought to be involved in regulating the hosts’ immune response. The proper biosynthesis of the myco-membrane is essential for the viability of CMN family pathogens, and the unique physiology of the myco-membrane has made it a primary target for pharmacological therapeutics. In addition to targeting peptidoglycan, the treatment of CMN family pathogens can also include the disruption of arabinogalactan polymerization and the synthesis of fatty acids used in the construction of the more complicated mycolic acids. Mycolic acids are long-chain saturated fatty acids unique to the CMN family and are found exclusively in the outer myco-membrane. Significant efforts have gone into documenting the biosynthetic pathway of mycolic acid, where it was revealed that the catalytic steps necessary to form these long-chain fatty acids are completed inside the bacterial cell. This raised the question of how these mycolic acids cross the cell membrane in order to be incorporated in the outer membrane. In M. tuberculosis it was shown that mycolic acids could not translocate to the outer leaflet of the inner membrane without the function of the essential membrane transporter MmpL3. The role of MmpL3 as a mycolic acid transporter has since been validated, and MmpL3 has been pursued as a novel pharmacological target. Despite the significance of this discovery, the exact mechanism by which MmpL3 transports mycolic acids across the cell membrane had not yet been described. The focus of this thesis is on the mechanistic function of the essential mycolic acid membrane transporters CmpL1 from C. glutamicum and MmpL3 from M. tuberculosis, their interactions with inhibitors, and the discovery of novel protein-MmpL3 interactions. To begin our study, we undertook the purification of MmpL3 from M. tuberculosis and CmpL1 from C. glutamicum, and reconstituted them into functional proteoliposomes. Our results from this experiment demonstrate that the mycolic acid transporters facilitate H+ flux across the membrane which is stimulated in the presence of a substrate analog. As MmpL3 is a significant therapeutic target, several MmpL3 inhibitors have been developed and are in clinical trials. Therefore, we next moved to determine whether these inhibitors directly interact with MmpL3, and if they carry out their function through the inhibition of the observed H+ transport. We utilized a Surface Plasmon Resonance (SPR) assay to successfully determine the direct interaction of a variety of pharmacophores, and we report the kinetic rates of the interactions. Continuing the study of mycolic acid transport inhibition, we pursued a set of novel indole-2-carboxamide (IC) compounds which had both direct interaction and good activity in minimum inhibitory concentration (MIC) assays against M. tuberculosis MmpL3 and C. glutamicum CmpL1. We utilized these IC inhibitors in the proteoliposome assays and report our results that the inhibition of mycolic acid transport is due to the inhibition of H+ flux across the membrane. MmpL3 carries out the essential function of transporting mycolic acids across the plasma membrane of the bacterial cell. For proper cell wall biogenesis, the timing and incorporation of mycolic acid into the nascent cell wall would need to be coordinated with other biosynthetic machinery. We hypothesized that there would need to be other proteins associated to MmpL3. To test this hypothesis, we identified potential protein interactions via a bacterial two-hybrid screen of the entire Mtb proteome with MmpL3. Potential interacting proteins were independently purified and assayed for direct interaction with MmpL3 using SPR, and we report our results confirming high affinity interactions with several proteins. This significant result provided the first description of the MmpL3 interactome

A systems approach to understanding the connection between farm systems resilience and pasture resilience

Farm systems resilience in New Zealand pasture-based farming is influenced by external drivers such as environmental regulation, and internal drivers such as existence, expressed as profitability. We examine ten published case studies of farm systems change to provide insight into management interventions to these drivers and their impacts on pasture resilience. Nutrient supply was key to increasing pasture longevity, water use efficiency and animal feed supply. Manipulating water use efficiency through irrigation and legume (predominantly lucerne) use increased nitrogen use efficiency and added pasture supply for animal consumption. Monitoring the pasture supply and animal response ensured both animal feed requirements and pasture conditions for future growth were met. The resilience of pastures was improved when monitoring guided adaptive management application to ensure whole-farm resilience

Habitat Use by Juvenile Gag, Mycteroperca microlepis (Pisces: Serranidae), in Subtropical Charlotte Harbor, Florida (USA)

Estuaries play a key role in the juvenile stage of gag (Myeteroperca microlepis). The use of estuarine habitats by juvenlle gag has been examined in temperate estuaries, which are at the northern limits of the range of this species, but the importance of subtropical estuaries during the early life history of this species has not been studied extensively. Gag were collected in subtropical Charlotte Harbor, Florida, during routine monthiy sampling from January 1996 to Decemher 2002. Juvenlle gag were collected using a 21.3-m seine, a 183-m haul seine, and a 183-m purse seine. A total of 738 individuals ranging from 30 to 489 mm standard length (SL) were collected in 4,480 samples. Most gag (96%) were probably young-of-the-year (\u3c 288 mm SL). The majority of juveniles were collected in polyhaline Gasparilla and Pine Island sounds from April to December, with a few larger individuals captured year-round. The observed period of gag settlement was similar to thet reported in other subtropical and temperate estuaries, but gag in Charlotte Harbor remained in the estuary longer and egressed at a larger size than did gag in other estuaries. Relative abundance of juvenile gag within Charlotte Harbor was greatest on shallow seagrass shoals but was also high along flinging mangrove shorelines, which is a habitat not previously reported for gag

Ewe Body Condition: Does Farmer Practice Meet Research Guidelines?

A group of sheep farmers in the Southland region of New Zealand were surveyed in the winter of 2011 to determine their current winter management practices and ewe performance. This followed a 3 year Sustainable Farming Fund project to investigate if there was a relationship between maintaining an even ewe body condition score (BCS) during pregnancy and the incidence of vaginal prolapse (bearings). The program included a series of workshops to help farmers improve their ewe winter management and it became apparent that the actual winter feeding practice and ewe performance was relatively unknown. The research was done to document current farmer winter feeding and management practices and subsequent whole flock reproductive performance

Faecal sludge management and technology justice: promoting sustained and scalable solutions

In this paper we use Practical Action’s framework of Technology Justice and apply it to faecal sludge management. The framework analyses FSM from the perspectives of access, use and innovation. It encourages a wider systems-based approach to the issue. We illustrate the ideas by discussing how we are trying to create long-lasting change through a positive enabling environment in Bangladesh which encompasses empowerment of informal pit emptiers, engagement with Municipalities, and work to establish a new set of national guidelines

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture

Conformal Robotic Stereolithography

Additive manufacturing by layerwise photopolymerization, commonly called stereolithography (SLA), is attractive due to its high resolution and diversity of materials chemistry. However, traditional SLA methods are restricted to planar substrates and planar layers that are perpendicular to a single-axis build direction. Here, we present a robotic system that is capable of maskless layerwise photopolymerization on curved surfaces, enabling production of large-area conformal patterns and the construction of conformal freeform objects. The system comprises an industrial six-axis robot and a custom-built maskless projector end effector. Use of the system involves creating a mesh representation of the freeform substrate, generation of a triangulated toolpath with curved layers that represents the target object to be printed, precision mounting of the substrate in the robot workspace, and robotic photopatterning of the target object by coordinated motion of the robot and substrate. We demonstrate printing of conformal photopatterns on spheres of various sizes, and construction of miniature three-dimensional objects on spheres without requiring support features. Improvement of the motion accuracy and development of freeform toolpaths would enable construction of polymer objects that surpass the size and support structure constraints imparted by traditional SLA systems.American Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipNational Institute of Mental Health (U.S.) (University of Michigan Microfluidics in Biomedical Sciences Training Program. 5T32-EB005582)Singapore-MIT Alliance for Research and Technology (SMART