A Necropsy Study of Disease and Comorbidity Trends in Morbidity and Mortality in the Koala (Phascolarctos cinereus) in South-East Queensland, Australia.

Koalas are an iconic Australian marsupial undergoing precipitous population reduction in South-East Queensland from complex interacting threats. To investigate the causes of death and the interaction of comorbidities with demography in South-East Queensland koalas, a large scale, high-throughput prospective necropsy survey was conducted spanning 2013-2016. During this period, 519 necropsies were conducted in 155 young/subadult koalas, 235 mature, 119 old koalas and 10 of unknown age. Similar numbers of males and females were assessed. Trauma and infectious disease at were the most common single diagnoses. However, comorbidity was frequent, including multicentric infection or infectious disease in combination with trauma or senescence. Female koalas had proportionally more reproductive chlamydiosis compared to males in which the ocular and urinary systems were more commonly affected. Comorbidity and disease were strongly associated with poor body condition, and trauma was associated with good body condition. Animals affected by motor vehicle trauma were often in better body condition than those affected by animal attack, tree fall or other causes of trauma. This study identified a higher frequency of infections and comorbidity then previously reported, confirming the complex nature of interacting threats to the koala population.

Recording body temperature in koalas (Phascolarctos cinereus): a comparison of techniques.

OBJECTIVE: Compare the use of four techniques to measure body temperature in koalas: intraperitoneal (thermal data logger and temperature sensitive radio transmitter), rectal (certified thermometer), tympanic (infrared thermometer), and hind foot (infrared camera). METHODS: The body temperature data collected concurrently from the intraperitoneal loggers were used as the benchmark in the analyses. RESULTS: The rectal, foot and tympanic methods consistently recorded lower body temperature when compared with the benchmark. There was a strong positive relationship (R2 = 0.79) between logger and rectal measurements, but no significant relationship between logger and foot or logger and tympanic measurements. CONCLUSION: Rectal measurements can be used to record internal body temperature, with the caveat that such measurements will generally register a temperature approximately 0.25°C lower than the actual intraperitoneal temperature.

First report of a spermatic granuloma and varicocele in a marsupial: A Koala (Phascolarctos cinereus) Case Study.

This study reports the first documented clinical case of a spermatic granuloma and varicocele in a marsupial. Initial clinical presentation included gross morphological changes in the left scrotal cord, epididymis and testis. Ultrasonography of the scrotum and spermatic cord, and gross and histopathological examination after hemicastration, confirmed the condition as a spermatic granuloma affecting the left caput epididymis, with a varicocele in the left proximal spermatic cord, which was causing azoospermia and infertility. Semen quality and serum testosterone secretion following a GnRH challenge was assessed prior to, and following surgery. After hemi-castration, an increase in androgen secretion to within normal reference ranges for the koala was observed with a subsequent increase in semen production and sperm quality resulting in the sire of a pouch young, 12months later.

Surgical implantation of temperature-sensitive transmitters and data-loggers to record body temperature in koalas (Phascolarctos cinereus).

BACKGROUND: Under predicted climate change scenarios, koala distribution in Australia is expected to be adversely affected. Recent studies have attempted to identify suitable habitat, based on models of bioclimatic regions, but to more accurately reflect the thermal tolerance and behavioural adaptations of the various regional populations, the koala's response to periods of heat stress will need to be investigated at the individual animal level. OBJECTIVE: To explore the safety and suitability of temperature-sensitive intra-abdominal implants for monitoring core body temperature in the koala. METHODS: A temperature-sensitive radio transmitter and thermal iButton data-logger, waxed together as a package, were surgically implanted into the abdominal cavity of four captive koalas. In one animal the implant was tethered and in the other three, it was left free-floating. RESULTS: After 3 months, the implants were removed and all four koalas recovered without complications. The tethering of the package in the one koala resulted in minor inflammation and adhesion, so this practice was subsequently abandoned. The free-floating deployments were complication-free and revealed a diurnal body temperature rhythm, with daily ranges of 0.4-2.8°C. The minimum recorded body temperature was 34.2°C and the maximum was 37.7°C. The difference in the readings obtained from the transmitters and iButtons never exceeded 0.3°C. CONCLUSIONS: The suitability of the surgical approach was confirmed, from both the animal welfare and data collection points of view.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction.

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway.

Human natural killer cells mediate killing of intracellular Mycobacterium tuberculosis H37Rv via granule-independent mechanisms.

Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogen Mycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellular M. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.

Arabidopsis thaliana RNA polymerase II subunits related to yeast and human RPB5.

Arabidopsis thaliana contains at least four genes that are predicted to encode polypeptides related to the RPB5 subunit found in yeast and human RNA polymerase II. This subunit has been shown to be the largest subunit common to yeast RNA polymerases I, II, and III (RPABC27). More than one of these genes is expressed in Arabidopsis suspension culture cells, but only one of the encoded polypeptides is found in purified RNA polymerases II and III. This polypeptide has a predicted pI of 9.6, matches 14 of 16 amino acids in the amino terminus of cauliflower RPB5 that was microsequenced, and shows 42 and 53% amino acid sequence identity with the yeast and human RPB5 subunits, respectively.

Mechanisms of Action and Dose-Response Relationships Governing Biological Control of Fusarium Wilt of Tomato by Nonpathogenic Fusarium spp.

ABSTRACT Three isolates of nonpathogenic Fusarium spp. (CS-1, CS-20, and Fo47), previously shown to reduce the incidence of Fusarium wilt diseases of multiple crops, were evaluated to determine their mechanisms of action and antagonist-pathogen inoculum density relationships. Competition for nutrients, as represented by a reduction in pathogen saprophytic growth in the presence of the biocontrol isolates, was observed to be an important mechanism of action for isolate Fo47, but not for isolates CS-1 and CS-20. All three biocontrol isolates demonstrated some degree of induced systemic resistance in tomato (Lycopersicon esculentum) and watermelon (Citrullus lanatus) plants, as determined by split-root tests, but varied in their relative abilities to reduce disease. Isolate CS-20 provided the most effective control (39 to 53% disease reduction), while Fo47 provided the least effective control (23 to 25% reduction) in split-root tests. Dose-response relationships also differed considerably among the three biocon-trol isolates, with CS-20 significantly reducing disease incidence at antagonist doses as low as 100 chlamydospores per g of soil (cgs) and at pathogen densities up to 10(5) cgs. Isolate CS-1 also was generally effective at antagonist densities of 100 to 5,000 cgs, but only when pathogen densities were below 10(4) cgs. Isolate Fo47 was effective only at antagonist densities of 10(4) to 10(5) cgs, regardless of pathogen density. Epidemiological dose-response models (described by linear, negative exponential, hyperbolic saturation [HS], and logistic [LG] functions) fit to the observed data were used to quantify differences among the biocontrol isolates and establish biocontrol characteristics. Each isolate required a different model to best describe its dose-response characteristics, with the HS/HS, LG/HS, and LG/LG models (pathogen/biocontrol components) providing the best fit for isolates CS-1, CS-20, and Fo47, respectively. Model parameters (defining effective biocontrol dose (ED(50)) indicated an ED(50) of 2.6, 36.3, and 2.1 x 10(6) cgs and estimates of biocontrol efficiency of 0.229, 0.539, and 0.774 for isolates CS-1, CS-20, and Fo47, respectively. Differences in dose-response relationships among the biocontrol isolates were attributed to differences in their mechanisms of action, with CS-20 and CS-1 functioning primarily by induced resistance and Fo47 functioning primarily by competition for nutrients.

Two small subunits in Arabidopsis RNA polymerase II are related to yeast RPB4 and RPB7 and interact with one another.

An Arabidopsis cDNA (AtRPB15.9) that encoded a protein related to the RPB4 subunit in yeast RNA polymerase II was cloned. The predicted molecular mass of 15.9 kDa for the AtRPB15.9 protein was significantly smaller than 25 kDa for yeast RBP4. In SDS-PAGE, AtRPB15.9 migrated as the seventh or eighth largest subunit (i.e. apparent molecular mass of 14-15 kDa) in Arabidopsis RNA polymerase II, whereas RPB4 migrates as the fourth largest subunit (i.e. apparent molecular mass of 32 kDa) in yeast RNA polymerase II. Unlike yeast RPB4 and RPB7, which dissociate from RNA polymerase II under mildly denaturing conditions, plant subunits related to RPB4 and RPB7 are more stably associated with the enzyme. Recombinant AtRPB15.9 formed stable complexes with AtRPB19.5 (i.e. a subunit related to yeast RPB7) in vitro as did recombinant yeast RPB4 and RPB7 subunits. Stable heterodimers were also formed between AtRPB15. 9 and yeast RPB7 and between yeast RPB4 and AtRPB19.5.

A Formulation of Trichoderma and Gliocladium to Reduce Damping-off Caused by Rhizoctonia solani and Saprophytic Growth of the Pathogen in Soilless Mix.

Commercially manufactured cellulose granules (Biodac) were mixed with a sticker and fermentor-produced biomass of isolates of Trichoderma spp. and Gliocladium virens to produce a formulation in which chlamydospores in the biomass were "activated" with dilute acid. Activation resulted in the formation of young, actively growing hyphae of the biocontrol fungi within a 2- to 3-day period under no special aseptic conditions. Activated Biodac with biomass of isolates Gl-3, Gl-21, and Gl-32 of G. virens and isolate TRI-4 of T. hamatum applied to soilless mix at a rate of 1.5% (wt/wt) reduced damping-off of eggplant caused by Rhizoctonia solani (R-23) and resulted in stands comparable to that (88%) in noninfested soilless mix. Saprophytic growth of the pathogen was also reduced. The application of either of two activated Biodac formulations to provide the same amount (1.5% with 9.4 mg of biomass per g of Biodac or 0.2% with 75.0 mg of biomass per g of Biodac) reduced preemergence damping-off as well as saprophytic growth of R-23. Also, there was about a 103-fold population increase of Gl-3 and TRI-4 in the soilless mix at the time of plant harvest compared with that provided to the soilless mix at the time of formulation addition. Activated Biodac of Gl-3 also reduced the spread of R-23 in soilless mix when the pathogen was applied at specific foci rather than evenly distributed. The inhibition of pathogen spread significantly reduced the postemergence damping-off of cucumber, eggplant, and pepper seedlings.

Reconstitution of yeast and Arabidopsis RNA polymerase alpha-like subunit heterodimers.

Two subunits of about 36-44 kDa and 13-19 kDa in the eukaryotic nuclear RNA polymerases share limited amino acid sequence similarity to the alpha subunit in Escherichia coli RNA polymerase. The alpha subunit in the prokaryotic enzyme has a stoichiometry of 2, but the stoichiometry of the alpha-like subunits in the eukaryotic enzymes is not entirely clear. To gain insight into the subunit stoichiometry and assembly pathway for eukaryotic RNA polymerases, in vitro reconstitution experiments have been carried out with recombinant alpha-like subunits from yeast and plant RNA polymerase II. The large and small alpha-like subunits from each species formed stable heterodimers in vitro, but neither the large or small alpha-like subunits formed stable homodimers. Furthermore, mixed heterodimers were formed between corresponding subunits of yeast and plants, but were not formed between corresponding subunits in different RNA polymerases from the same species. Our results suggest that RNA polymerase II alpha-like heterodimers may be the equivalent of alpha homodimers found in E. coli RNA polymerase.

A 14-kDa Arabidopsis thaliana RNA polymerase III subunit contains two alpha-motifs flanked by a highly charged C terminus.

We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19. Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13-15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At. The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit. AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the alpha subunit of Escherichia coli (Ec) Pol. AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16.

Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II.

Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the alpha subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic alpha subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II.

Arabidopsis expresses two genes that encode polypeptides similar to the yeast RNA polymerase I and III AC40 subunit.

A 40-kDa subunit in eukaryotic RNA polymerases (Pol) I and III (e.g., yeast yAC40) is related in a part of its aa sequence to the alpha subunit of prokaryotic Pol and to a 35-44-kDa subunit in Pol II (e.g., yeast yB44). We have cloned two cDNAs, AtRPAC42 and AtRPAC43, from an Arabidopsis thaliana (At) (ecotype Columbia) lambda Yes expression library that encode Pol I and III subunits related to yAC40. The aa sequences derived from the cDNA clones were found to be 72% identical to each other and 40% identical to yeast Pol I and III subunits yAC40, but only 30% identical to yeast Pol II subunit yB44. While most other nuclear Pol genes identified to date are single-copy genes, two genes encode 42 and 43-kDa subunits of At Pol I and/or III. A 42-kDa subunit with identical mobility in SDS-PAGE to the aAC42 in vitro translated subunit is found in Pol III purified from At suspension culture cells.

Use of 3D reconstruction to correct for patient motion in SPECT.

Patient motion occurring during data acquisition in single photon emission computed tomography (SPET) can cause serious reconstruction artefacts. We have developed a new approach to correct for head motion in brain SPECT. Prior to motion, projections are assigned to conventional projections. When head motion occurs, it is measured by a motion monitoring system, and subsequent projection data are mapped to 'virtual' projections. The appropriate position of each virtual projection is determined by applying the converse of the patient's accumulated motion to the actual camera projection. Conventional and virtual projections, taken together, form a consistent set that can be reconstructed using a three-dimensional (3D) algorithm. The technique has been tested on a range of simulated rotational movements, both within and out of the transaxial plane. For all simulated movements, the motion corrected images exhibited better agreement with a motion free reconstruction than did the uncorrected images. This technique may help to overcome one of the major remaining limitations on image quality and quantitative accuracy in SPECT.

Accelerated image reconstruction using ordered subsets of projection data.

The authors define ordered subset processing for standard algorithms (such as expectation maximization, EM) for image restoration from projections. Ordered subsets methods group projection data into an ordered sequence of subsets (or blocks). An iteration of ordered subsets EM is defined as a single pass through all the subsets, in each subset using the current estimate to initialize application of EM with that data subset. This approach is similar in concept to block-Kaczmarz methods introduced by Eggermont et al. (1981) for iterative reconstruction. Simultaneous iterative reconstruction (SIRT) and multiplicative algebraic reconstruction (MART) techniques are well known special cases. Ordered subsets EM (OS-EM) provides a restoration imposing a natural positivity condition and with close links to the EM algorithm. OS-EM is applicable in both single photon (SPECT) and positron emission tomography (PET). In simulation studies in SPECT, the OS-EM algorithm provides an order-of-magnitude acceleration over EM, with restoration quality maintained.