Unravelling the Diversity of Microorganisms in Ticks from Australian Wildlife.

Ticks and tick-borne pathogens pose a significant threat to the health and welfare of humans and animals. Our knowledge about pathogens carried by ticks of Australian wildlife is limited. This study aimed to characterise ticks and tick-borne microorganisms from a range of wildlife species across six sites in Victoria, Australia. Following morphological and molecular characterisation (targeting 16S rRNA and cytochrome c oxidase I), tick DNA extracts (n = 140) were subjected to microfluidic real-time PCR-based screening for the detection of microorganisms and Rickettsia-specific real-time qPCRs. Five species of ixodid ticks were identified, including Aponomma auruginans, Ixodes (I.) antechini, I. kohlsi, I. tasmani and I. trichosuri. Phylogenetic analyses of 16S rRNA sequences of I. tasmani revealed two subclades, indicating a potential cryptic species. The microfluidic real-time PCR detected seven different microorganisms as a single (in 13/45 ticks) or multiple infections (27/45). The most common microorganisms detected were Apicomplexa (84.4%, 38/45) followed by Rickettsia sp. (55.6%, 25/45), Theileria sp. (22.2% 10/45), Bartonella sp. (17.8%, 8/45), Coxiella-like sp. (6.7%, 3/45), Hepatozoon sp. (2.2%, 1/45), and Ehrlichia sp. (2.2%, 1/45). Phylogenetic analyses of four Rickettsia loci showed that the Rickettsia isolates detected herein potentially belonged to a novel species of Rickettsia. This study demonstrated that ticks of Australian wildlife carry a diverse array of microorganisms. Given the direct and indirect human-wildlife-livestock interactions, there is a need to adopt a One Health approach for continuous surveillance of tick-associated pathogens/microorganisms to minimise the associated threats to animal and human health.

Maternal transmission of an Igf2r domain 11: IGF2 binding mutant allele (Igf2rI1565A) results in partial lethality, overgrowth and intestinal adenoma progression.

The cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R or IGF2R) traffics IGF2 and M6P ligands between pre-lysosomal and extra-cellular compartments. Specific IGF2 and M6P high-affinity binding occurs via domain-11 and domains-3-5-9, respectively. Mammalian maternal Igf2r allele expression exceeds the paternal allele due to imprinting (silencing). Igf2r null-allele maternal transmission results in placenta and heart over-growth and perinatal lethality (>90%) due to raised extra-cellular IGF2 secondary to impaired ligand clearance. It remains unknown if the phenotype is due to either ligand alone, or to both ligands. Here, we evaluate Igf2r specific loss-of-function of the domain-11 IGF2 binding site by replacing isoleucine with alanine in the CD loop (exon 34, I1565A), a mutation also detected in cancers. Igf2rI1565A/+p maternal transmission (heterozygote), resulted in placental and embryonic over-growth with reduced neonatal lethality (<60%), and long-term survival. The perinatal mortality (>80%) observed in homozygotes (Igf2rI1565A/I1565A) suggested that wild-type paternal allele expression attenuates the heterozygote phenotype. To evaluate Igf2r tumour suppressor function, we utilised intestinal adenoma models known to be Igf2 dependent. Bi-allelic Igf2r expression suppressed intestinal adenoma (ApcMin). Igf2rI1565A/+p in a conditional model (Lgr5-Cre, Apcloxp/loxp) resulted in worse survival and increased adenoma proliferation. Growth, survival and intestinal adenoma appear dependent on IGF2R-domain-11 IGF2 binding.

Regeneration of Dopaminergic Neurons in Adult Zebrafish Depends on Immune System Activation and Differs for Distinct Populations.

Adult zebrafish, in contrast to mammals, regenerate neurons in their brain, but the extent and variability of this capacity is unclear. Here we ask whether the loss of various dopaminergic neuron populations is sufficient to trigger their functional regeneration. Both sexes of zebrafish were analyzed. Genetic lineage tracing shows that specific diencephalic ependymo-radial glial (ERG) progenitor cells give rise to new dopaminergic [tyrosine hydroxylase-positive (TH+)] neurons. Ablation elicits an immune response, increased proliferation of ERG progenitor cells, and increased addition of new TH+ neurons in populations that constitutively add new neurons (e.g., diencephalic population 5/6). Inhibiting the immune response attenuates neurogenesis to control levels. Boosting the immune response enhances ERG proliferation, but not addition of TH+ neurons. In contrast, in populations in which constitutive neurogenesis is undetectable (e.g., the posterior tuberculum and locus ceruleus), cell replacement and tissue integration are incomplete and transient. This is associated with a loss of spinal TH+ axons, as well as permanent deficits in shoaling and reproductive behavior. Hence, dopaminergic neuron populations in the adult zebrafish brain show vast differences in regenerative capacity that correlate with constitutive addition of neurons and depend on immune system activation.SIGNIFICANCE STATEMENT Despite the fact that zebrafish show a high propensity to regenerate neurons in the brain, this study reveals that not all types of dopaminergic neurons are functionally regenerated after specific ablation. Hence, in the same adult vertebrate brain, mechanisms of successful and incomplete regeneration can be studied. We identify progenitor cells for dopaminergic neurons and show that activating the immune system promotes the proliferation of these cells. However, in some areas of the brain this only leads to insufficient replacement of functionally important dopaminergic neurons that later disappear. Understanding the mechanisms of regeneration in zebrafish may inform interventions targeting the regeneration of functionally important neurons, such as dopaminergic neurons, from endogenous progenitor cells in nonregenerating mammals.

A synthetic cell permeable antioxidant protects neurons against acute oxidative stress.

Excessive reactive oxygen species (ROS) can damage proteins, lipids, and DNA, which result in cell damage and death. The outcomes can be acute, as seen in stroke, or more chronic as observed in age-related diseases such as Parkinson's disease. Here we investigate the antioxidant ability of a novel synthetic flavonoid, Proxison (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), using a range of in vitro and in vivo approaches. We show that, while it has radical scavenging ability on par with other flavonoids in a cell-free system, Proxison is orders of magnitude more potent than natural flavonoids at protecting neural cells against oxidative stress and is capable of rescuing damaged cells. The unique combination of a lipophilic hydrocarbon tail with a modified polyphenolic head group promotes efficient cellular uptake and moderate mitochondrial enrichment of Proxison. Importantly, in vivo administration of Proxison demonstrated effective and well tolerated neuroprotection against cell loss in a zebrafish model of dopaminergic neurodegeneration.

Cleaving the Halqeh-ye-nur diamonds: a dynamic fracture analysis.

The degree of surface roughness and clarity with which a surface in a brittle material can be formed via fracture is known to be related to the speed of the propagating crack. Cracks traversing a brittle material at low speed produce very smooth surfaces, while those propagating faster create less reflective and rough surfaces (Buehler MJ, Gao H. 2006 Nature 439, 307-310 (doi:10.1038/nature04408)). The elastic wave speeds (c(l)≈18 000 m s(-1), c(s)≈11 750 m s(-1)) in diamond are fast (Willmott GR, Field JE. 2006 Phil. Mag. 86, 4305-4318 (doi:10.1080/14786430500482336)) and present a particular problem in creating smooth surfaces during the cleaving of diamond-a routine operation in the fashioning of diamonds for gemstone purposes--as the waves are reflected from the boundaries of the material and can add a tensile component to the propagating crack tip causing the well-known cleavage steps observed on diamond surfaces (Field JE. 1971 Contemp. Phys. 12, 1-31 (doi:10.1080/00107517108205103); Field JE. 1979 Properties of diamond, 1st edn, Academic Press; Wilks EM. 1958 Phil. Mag. 3, 1074-1080 (doi:10.1080/14786435808237036)). Here we report an analysis of two diamonds, having large dimensions and high aspect ratio, which from a gemological analysis are shown to have been cleaved from the same 200 carat specimen. A methodology for their manufacture is calculated by an analysis of a model problem. This takes into account the effect of multiple reflections from the sample boundaries. It is suggested that the lapidary had an intuitive guide to how to apply the cleavage force in order to control the crack speed. In particular, it is shown that it is likely that this technique caused the fracture to propagate at a lower speed. The sacrifice of a large diamond with the intention of creating thin plates, rather than a faceted gemstone, demonstrates how symbolism and beliefs associated with gemstones have changed over the centuries (Harlow GE. 1998 The nature of diamonds, Cambridge University Press). The scientific insights gained by studying these gemstones suggest a method of producing macroscale atomically flat and stress-free surfaces on other brittle materials.

Reporting for duty: the paternal function and clinical formulations.

The author highlights some developments in the theory of the preoedipal paternal function and paternal functionary and incorporates these ideas in developing clinical formulations for four clinical cases that privilege the preoedipal paternal function. In particular, four aspects of the preoedipal paternal function are identified, and for each a clinical case is discussed. Emphasis is placed on the necessity of widening clinical formulations to ensure clinicians have the widest possible set of clinical ideas and hence interventions and techniques at their fingertips.

ANO5 gene analysis in a large cohort of patients with anoctaminopathy: confirmation of male prevalence and high occurrence of the common exon 5 gene mutation.

Limb girdle muscular dystrophy type 2L or anoctaminopathy is a condition mainly characterized by adult onset proximal lower limb muscular weakness and raised CK values, due to recessive ANO5 gene mutations. An exon 5 founder mutation (c.191dupA) has been identified in most of the British and German LGMD2L patients so far reported. We aimed to further investigate the prevalence and spectrum of ANO5 gene mutations and related clinical phenotypes, by screening 205 undiagnosed patients referred to our molecular service with a clinical suspicion of anoctaminopathy. A total of 42 unrelated patients had two ANO5 mutations (21%), whereas 14 carried a single change. We identified 34 pathogenic changes, 15 of which are novel. The c.191dupA mutation represents 61% of mutated alleles and appears to be less prevalent in non-Northern European populations. Retrospective clinical analysis corroborates the prevalently proximal lower limb phenotype, the male predominance and absence of major cardiac or respiratory involvement. Identification of cases with isolated hyperCKaemia and very late symptomatic male and female subjects confirms the extension of the phenotypic spectrum of the disease. Anoctaminopathy appears to be one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of about 20%-25% in unselected undiagnosed cases.

Comparative outcome of oesophagogastric cancer in younger patients.

INTRODUCTION: The incidence of oesophageal and gastric cardia adenocarcinoma has increased rapidly over the previous two decades. There has been a rise in the number of younger patients affected, and the disease may be more aggressive and have a worse prognosis in these individuals. Current UK guidelines for urgent cancer referral focus on patients who are over 55 years. This study prospectively compares the referral times and outcome in a cohort of patients diagnosed with oesophagogastric cancer under the age of 55 years with a matched cohort over 55 of age. PATIENTS AND METHODS: Every patient diagnosed with oesophageal, junctional or gastric cancer under the age of 55 years and every subsequent patient over the age of 55 years was accepted into this study. In all, 17 hospitals participated over a 12-month period. The following data were recorded: duration of symptoms, number of fast-track referrals, duration from GP referral to first hospital visit and stage at presentation. A survival analysis between the two groups was conducted at 2 years after the end of recruitment. RESULTS: In total, 102 patients under the age of 55 years were diagnosed with oesophagogastric cancer during the study period. There were fewer fast-track referrals from GPs in this group compared to the over 55-year matched cases (29.4% vs 40.2%). Duration of time from GP referral to first hospital visit was significantly longer in the under 55-year group (median 14 days vs 11 days; P = 0.045 Mann-Whitney). Stage at presentation was similar between groups, but a higher proportion of patients under 55 years were offered a curative treatment plan compared to those over 55 years (P < 0.01). Survival analysis conducted at 2 years after the end of recruitment demonstrated a longer median survival in the under 55-year group (348 days vs 248 days; P = 0.03 log rank). CONCLUSIONS: Although there was a longer referral time in patients under the age of 55 years, this had no effect on disease stage at presentation. Patients under the age of 55 years diagnosed with oesophageal or gastric cancer appear to have a better prognosis than those aged over 55 years.

An unusual presentation of a Gastrointestinal stromal tumour (GIST).

BACKGROUND: Gastrointestinal stromal tumours (GIST) are rare tumours, now more frequently identified with the new imaging modalities like computerised tomography (CT) and magnetic resonance imaging (MRI). We report a rare presentation of a GIST with an unusual diagnostic workup in a multidisciplinary setting leading to a definitive diagnosis and treatment. CASE PRESENTATION: A 55-year-old lady was admitted under the general surgeons, with 3-day history of abdominal pain, three-week history of loss of appetite and weight. The patient was sequentially investigated with ultrasonography, computerised tomography and finally selective angiogram in a multidisciplinary setting. The selective angiogram showed a GIST with intratumour bleed, leading to successful surgical excision and being recurrence free at 22 month follow up. CONCLUSION: Clinical presentation of these tumours can be varied and gastrointestinal bleeding is the commonest mode described in the literature. The clinician needs to be aware of much more rare presentations of the GIST including an intra tumour bleed. A structured multidisciplinary approach would lead to successful diagnosis and treatment.

Gastrointestinal stromal tumors (GIST) and laparoscopic resection.

AIM: To review management, outcome and the lessons learnt from a laparoscopic approach to GISTs. METHOD: All cases of GIST presenting to the upper GI MDT between 2000 and 2006 were reviewed. Presentation, preoperative investigations, management and follow-up were recorded. Surgical resection using a laparoscopic approach, where feasible was the preferred management. RESULTS: 25 consecutive patients that included one oesophageal, three oesophago-gastric, 19 gastric and two smallbowel GISTs were treated between 2000 and 2006. There were 11 male and 14 females with a median age of 68 (25-90) years. Clinical presentation was: gastrointestinal bleed 15, pain 6, dysphagia 2, anaemia 3, weight loss 1, and asymptomatic 2. Out of 25, four were inoperable and treated with imatinib. 17 laparoscopic (including 2 conversions) and four open procedures were performed. Two (both GISTs close to the oesophago-gastric junction) required reoperation due to surgical-related morbidity. Of the 25, five were high-, 11 intermediate- and nine low-risk GISTs. No recurrences in follow-up (median 24, range 6-75) months was observed. CONCLUSION: GISTs can safely and effectively be treated laparoscopically although larger GISTs in difficult anatomical locations may require open surgery.

Tetherless fiber-coupled optical sources for extended metronomic photodynamic therapy.

BACKGROUND: Metronomic photodynamic therapy (mPDT) involves the delivery of a low fluence rate of light and photosensitizing drug, continuously and over an extended period. However, in mPDT trials, there has been a perceived need for light sources that can deliver light reliably for extended periods, while being small enough to be tolerated by small animals. METHODS: We report on the development of tetherless, lightweight, fiber-coupled optical sources for in vivo delivery of interstitial mPDT. Two forms are reported, based on diode lasers and light-emitting diodes (LEDs), the latter types weighing only 16.5g. RESULTS: The prototypes have been well tolerated in preliminary trials on tumor-bearing rat models and can currently provide stable levels of performance for upwards of 5 days. We also report an extension of the concept to units coupled to several optical fibers to enable simultaneous irradiation of multiple locations in tissue. CONCLUSION: These prototypes fulfill a current need for reliable mPDT optical sources for use with animal models. They also serve as the foundation for the development of fiber-coupled sources for use in future clinical trials.

Interstitial in vivo ALA-PpIX mediated metronomic photodynamic therapy (mPDT) using the CNS-1 astrocytoma with bioluminescence monitoring.

BACKGROUND: We report the first truly metronomic delivery of photodynamic therapy using the rat-derived CNS-1 astrocytoma, a model with close histopathology with human brain tumours. METHODS: Metronomic PDT (mPDT) was delivered to CNS-1 bearing female Lewis rats. 5-Aminoluvelinic acid was delivered continuously through drinking water, while light was delivered via tetherless, light-weight, LED-based fiber coupled optical sources. Tumour burden before and after mPDT treatment was determined using bioluminescence imaging (BLI). RESULTS: Preliminary studies demonstrated that 24h of continuous mPDT illumination was capable of destroying small tumours (7 days post-implant). The reduction or elimination of tumour was confirmed using BLI and corroborated by histology. Additional studies showed that 24 and 48h continuous mPDT illumination had the capability to delay tumour re-growth by a period corresponding to approximately two doubling times. Animals given 4-day mPDT did not show any signs of tumour re-growth via BLI at 26 days post-tumour implantation. CONCLUSIONS: In summary, these results demonstrate the feasibility of delivering mPDT for extended periods, as well as its potential as a treatment for small brain tumours.

No immediate pain relief for the pharmaceutical industry.

Over many years, the pharmaceutical industry has developed a highly successful model for making new medicines. Currently, there are an estimated 13,000 prescription drugs on the market. However, the model that has served the sector so well for so long is now breaking down. Lack of productivity in the laboratory, the imminent expiry of patents for numerous best selling products, intense competition and a more demanding marketplace, are all signs of a need for a new approach to drug discovery. Consequently, some pharmaceutical executives may feel that they are managing a previously prestigous team, which now find themselves in the relegation zone, and cannot continue under the same model. This article suggests that radical transformation of the business at every level is the only key to long-term success.

Bayesian variable selection in multinomial probit models to identify molecular signatures of disease stage.

Here we focus on discrimination problems where the number of predictors substantially exceeds the sample size and we propose a Bayesian variable selection approach to multinomial probit models. Our method makes use of mixture priors and Markov chain Monte Carlo techniques to select sets of variables that differ among the classes. We apply our methodology to a problem in functional genomics using gene expression profiling data. The aim of the analysis is to identify molecular signatures that characterize two different stages of rheumatoid arthritis.

Structure-activity relationships in purine-based inhibitor binding to HSP90 isoforms.

Inhibition of the ATPase activity of the chaperone protein HSP90 is a potential strategy for treatment of cancers. We have determined structures of the HSP90alpha N-terminal domain complexed with the purine-based inhibitor, PU3, and analogs with enhanced potency both in enzyme and cell-based assays. The compounds induce upregulation of HSP70 and downregulation of the known HSP90 client proteins Raf-1, CDK4, and ErbB2, confirming that the molecules inhibit cell growth by a mechanism dependent on HSP90 inhibition. We have also determined the first structure of the N-terminal domain of HSP90beta, complexed with PU3. The structures allow a detailed rationale to be developed for the observed affinity of the PU3 class of compounds for HSP90 and also provide a structural framework for design of compounds with improved binding affinity and drug-like properties.