A shift from cattle to camel and goat farming can sustain milk production with lower inputs and emissions in north sub-Saharan Africa's drylands.

Climate change is increasingly putting milk production from cattle-based dairy systems in north sub-Saharan Africa (NSSA) under stress, threatening livelihoods and food security. Here we combine livestock heat stress frequency, dry matter feed production and water accessibility data to understand where environmental changes in NSSA's drylands are jeopardizing cattle milk production. We show that environmental conditions worsened for ∼17% of the study area. Increasing goat and camel populations by ∼14% (∼7.7 million) and ∼10% (∼1.2 million), respectively, while reducing the dairy cattle population by ∼24% (∼5.9 million), could result in ∼0.14 Mt (+5.7%) higher milk production, lower water (-1,683.6 million m3, -15.3%) and feed resource (-404.3 Mt, -11.2%) demand-and lower dairy emissions by ∼1,224.6 MtCO2e (-7.9%). Shifting herd composition from cattle towards the inclusion of, or replacement with, goats and camels can secure milk production and support NSSA's dairy production resilience against climate change.

Design rules for efficient transgene expression in plants.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935- and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Optimal procedure planning and guidance system for peripheral bronchoscopy.

With the development of multidetector computed-tomography (MDCT) scanners and ultrathin bronchoscopes, the use of bronchoscopy for diagnosing peripheral lung-cancer nodules is becoming a viable option. The work flow for assessing lung cancer consists of two phases: 1) 3-D MDCT analysis and 2) live bronchoscopy. Unfortunately, the yield rates for peripheral bronchoscopy have been reported to be as low as 14%, and bronchoscopy performance varies considerably between physicians. Recently, proposed image-guided systems have shown promise for assisting with peripheral bronchoscopy. Yet, MDCT-based route planning to target sites has relied on tedious error-prone techniques. In addition, route planning tends not to incorporate known anatomical, device, and procedural constraints that impact a feasible route. Finally, existing systems do not effectively integrate MDCT-derived route information into the live guidance process. We propose a system that incorporates an automatic optimal route-planning method, which integrates known route constraints. Furthermore, our system offers a natural translation of the MDCT-based route plan into the live guidance strategy via MDCT/video data fusion. An image-based study demonstrates the route-planning method's functionality. Next, we present a prospective lung-cancer patient study in which our system achieved a successful navigation rate of 91% to target sites. Furthermore, when compared to a competing commercial system, our system enabled bronchoscopy over two airways deeper into the airway-tree periphery with a sample time that was nearly 2 min shorter on average. Finally, our system's ability to almost perfectly predict the depth of a bronchoscope's navigable route in advance represents a substantial benefit of optimal route planning.

Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane.

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally-controlled expression of a silencing-resistant gene encoding a vacuole-targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole-cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high-isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value-added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing-resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field-grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.

Computer-based route-definition system for peripheral bronchoscopy.

Multi-detector computed tomography (MDCT) scanners produce high-resolution images of the chest. Given a patient's MDCT scan, a physician can use an image-guided intervention system to first plan and later perform bronchoscopy to diagnostic sites situated deep in the lung periphery. An accurate definition of complete routes through the airway tree leading to the diagnostic sites, however, is vital for avoiding navigation errors during image-guided bronchoscopy. We present a system for the robust definition of complete airway routes suitable for image-guided bronchoscopy. The system incorporates both automatic and semiautomatic MDCT analysis methods for this purpose. Using an intuitive graphical user interface, the user invokes automatic analysis on a patient's MDCT scan to produce a series of preliminary routes. Next, the user visually inspects each route and quickly corrects the observed route defects using the built-in semiautomatic methods. Application of the system to a human study for the planning and guidance of peripheral bronchoscopy demonstrates the efficacy of the system.

Robust 3-D airway tree segmentation for image-guided peripheral bronchoscopy.

A vital task in the planning of peripheral bronchoscopy is the segmentation of the airway tree from a 3-D multidetector computed tomography chest scan. Unfortunately, existing methods typically do not sufficiently extract the necessary peripheral airways needed to plan a procedure. We present a robust method that draws upon both local and global information. The method begins with a conservative segmentation of the major airways. Follow-on stages then exhaustively search for additional candidate airway locations. Finally, a graph-based optimization method counterbalances both the benefit and cost of retaining candidate airway locations for the final segmentation. Results demonstrate that the proposed method typically extracts 2-3 more generations of airways than several other methods, and that the extracted airway trees enable image-guided bronchoscopy deeper into the human lung periphery than past studies.

Image-based reporting for bronchoscopy.

Bronchoscopy is often performed for staging lung cancer. The recent development of multidetector computed tomography (MDCT) scanners and ultrathin bronchoscopes now enable the bronchoscopic biopsy and treatment of peripheral diagnostic regions of interest (ROIs). Because these ROIs are often located several generations within the airway tree, careful planning and interpretation of the bronchoscopic route is required prior to a procedure. The current practice for planning bronchoscopic procedures, however, is difficult, error prone, and time consuming. To alleviate these issues, we propose a method for producing and previewing reports for bronchoscopic procedures using patient-specific MDCT chest scans. The reports provide quantitative data about the bronchoscopic routes and both static and dynamic previews of the proper airway route. The previews consist of virtual bronchoscopic endoluminal renderings along the route and three-dimensional cues for a final biopsy site. The reports require little storage space and computational resources, enabling physicians to view the reports on a portable tablet PC. To evaluate the efficacy of the reporting system, we have generated reports for 22 patients in a human lung cancer patient pilot study. For 17 of these patients, we used the reports in conjunction with live image-based bronchoscopic guidance to direct physicians to central chest and peripheral ROIs for subsequent diagnostic evaluation. Our experience shows that the tool enabled useful procedure preview and an effective means for planning strategy prior to a live bronchoscopy.

3D MDCT-based system for planning peripheral bronchoscopic procedures.

The diagnosis and staging of lung cancer often begins with the assessment of a suspect peripheral chest site. Such suspicious peripheral sites may be solitary pulmonary nodules or other abnormally appearing regions of interest (ROIs). The state-of-the-art process for assessing such peripheral ROIs involves off-line procedure planning using a three-dimensional (3D) multidetector computed tomography (MDCT) chest scan followed by bronchoscopy with an ultrathin bronchoscope. We present an integrated computer-based system for planning peripheral bronchoscopic procedures. The system takes a 3D MDCT chest image as input and performs nearly all operations automatically. The only interaction required by the physician is the selection of ROI locations. The system is computationally efficient and fits smoothly within the clinical work flow. Integrated into the system and described in detail in the paper is a new surface-definition method, which is vital for effective analysis and planning to peripheral sites. Results demonstrate the efficacy of the system and its usage for the live guidance of ultrathin bronchoscopy to the periphery.

Host-delivered RNAi: an effective strategy to silence genes in plant parasitic nematodes.

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Acute bidirectional manipulation of muscle glucose uptake by in vivo electrotransfer of constructs targeting glucose transporter genes.

Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 +/- 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism.

Simple, robust strategies for generating DNA-directed RNA interference constructs.

We describe two complementary strategies for preparing DNA-directed RNA interference (ddRNAi) constructs designed to express hpRNA. The first, oligonucleotide assembly (OA), uses a very simple annealing protocol to combine up to 20 short nucleotides. These are then cloned into appropriately designed restriction sites in expression vectors. OA can be used to prepare simple hairpin (hp)-expressing constructs, but we prefer to use the approach to generate longer constructs. The second strategy, long-range cloning (LRC), uses a novel adaptation of long-range PCR protocols. For LRC, entire vectors are amplified with primers that serve to introduce short sequences into plasmids at defined anchor sites during PCR. The LCR strategy has proven highly reliable in our hands for generating simple ddRNAi constructs. Moreover, LCR is likely to prove useful in many situations in which conventional cloning strategies might prove problematic. In combination, OA and LRC can greatly simplify the design and generation of many expression constructs, including constructs for ddRNAi.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart.

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA(3) treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.

Randomly Amplified DNA Fingerprinting: A Culmination of DNA Marker Technologies Based on Arbitrarily-Primed PCR Amplification.

Arbitrarily-primed DNA markers can be very useful for genetic fingerprinting and for facilitating positional cloning of genes. This class of technologies is particularly important for less studied species, for which genome sequence information is generally not known. The technologies include Randomly Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting (DAF), and Amplified Fragment Length Polymorphism (AFLP). We have modified the DAF protocol to produce a robust PCR-based DNA marker technology called Randomly Amplified DNA Fingerprinting (RAF). While the protocol most closely resembles DAF, it is much more robust and sensitive because amplicons are labelled with either radioactive 33P or fluorescence in a 30-cycle PCR, and then separated and detected on large polyacrylamide sequencing gels. Highly reproducible RAF markers were readily amplified from either purified DNA or alkali-treated intact leaf tissue. RAF markers typically display dominant inheritance. However, a small but significant portion of the RAF markers exhibit codominant inheritance and represent microsatellite loci. RAF compares favorably with AFLP for efficiency and reliability on many plant genomes, including the very large and complex genomes of sugarcane and wheat. While the two technologies detect about the same number of markers per large polyacrylamide gel, advantages of RAF over AFLP include: (i) no requirement for enzymatic template preparation, (ii) one instead of two PCRs, and (iii) overall cost. RAF and AFLP were shown to differ in the selective basis of amplification of markers from genomes and could therefore be used in complementary fashion for some genetic studies.