Histopathology of a threatened surf clam, toheroa (Paphies ventricosa) from Aotearoa New Zealand.

The toheroa (Paphies ventricosa) is endemic to Aotearoa (New Zealand). Following decades of overfishing in the 1900 s, commercial and recreational fishing of toheroa is now prohibited. For unknown reasons, protective measures in place for over 40 years have not ensured the recovery of toheroa populations. For the first time, a systematic pathology survey was undertaken to provide a baseline of toheroa health in remaining major populations. Using histopathology, parasites and pathologies in a range of tissues are assessed and quantified spatio-temporally. Particular focus is placed on intracellular microcolonies of bacteria (IMCs). Bayesian ordinal logistic regression is used to model IMC infection and several facets of toheroa health. Model outputs show condition to be the most important predictor of IMC intensity in toheroa tissues. The precarious state of many toheroa populations around Aotearoa should warrant greater attention from scientists, conservationists, and regulators. It is hoped that this study will provide some insight into the current health status of a treasured and iconic constituent of several expansive surf beaches in Aotearoa.

Enigmatic MELK: The controversy surrounding its complex role in cancer.

The Ser/Thr protein kinase MELK (maternal embryonic leucine zipper kinase) has been considered an attractive therapeutic target for managing cancer since 2005. Studies using expression analysis have indicated that MELK expression is higher in numerous cancer cells and tissues than in their normal, nonneoplastic counterparts. Further, RNAi-mediated MELK depletion impairs proliferation of multiple cancers, including triple-negative breast cancer (TNBC), and these growth defects can be rescued with exogenous WT MELK, but not kinase-dead MELK complementation. Pharmacological MELK inhibition with OTS167 (alternatively called OTSSP167) and NVS-MELK8a, among other small molecules, also impairs cancer cell growth. These collective results led to MELK being classified as essential for cancer proliferation. More recently, in 2017, the proliferation of TNBC and other cancer cell lines was reported to be unaffected by genetic CRISPR/Cas9-mediated MELK deletion, calling into question the essentiality of this kinase in cancer. To date, the requirement of MELK in cancer remains controversial, and mechanisms underlying the disparate growth effects observed with RNAi, pharmacological inhibition, and CRISPR remain unclear. Our objective with this review is to highlight the evidence on both sides of this controversy, to provide commentary on the purported requirement of MELK in cancer, and to emphasize the need for continued elucidation of the functions of MELK.

Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.

Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue.

Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits an olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, we determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. We also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed.

Characterisation and distribution of the bacterial genus Endozoicomonas in a threatened surf clam.

The toheroa Paphies ventricosa is a large Aotearoa New Zealand (ANZ) endemic surf clam of cultural importance to many Maori, the Indigenous people of ANZ. Extensive commercial and recreational harvesting in the 20th century dramatically reduced populations, leading to the collapse and closure of the fishery. Despite being protected for >40 yr, toheroa have inexplicably failed to recover. In 2017, intracellular microcolonies (IMCs) of bacteria were detected in 'sick' toheroa in northern ANZ. Numerous mass mortality events (MMEs) have recently been recorded in ANZ shellfish, with many events linked by the presence of IMCs resembling Rickettsia-like organisms (RLOs). While similar IMCs have been implicated in MMEs in surf clams elsewhere, the impact of these IMCs on the health or recovery of toheroa is unknown. A critical first step towards understanding the significance of a pathogen in a host population is pathogen identification and characterisation. To begin this process, we examined 16S rRNA gene sequences of the putative IMCs from 4 toheroa populations that showed 97% homology to Endozoicomonas spp. sequences held in GenBank. Phylogenetic analysis identified closely related Endozoicomonas strains from the North and South Island, ANZ, and in situ hybridization, using 16S rRNA gene probes, confirmed the presence of the sequenced IMC gene in the gill and digestive gland tissues of toheroa. Quantitative PCR revealed site-specific and seasonal abundance patterns of Endozoicomonas spp. in toheroa populations. Although implicated in disease outbreaks elsewhere, the role of Endozoicomonas spp. within the ANZ shellfish mortality landscape remains uncertain.

Kinome Profiling Identifies Druggable Targets for Novel Human Cytomegalovirus (HCMV) Antivirals.

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of <1 µm, and at doses that were not toxic to uninfected cells. The most potent inhibitor of HCMV replication was OTSSP167 (IC50 <1.2 nm), a MELK inhibitor, blocked HCMV early gene expression and viral DNA accumulation, resulting in a >3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.

Measuring Kinase Activity-A Global Challenge.

The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc.

The Chthonomonas calidirosea Genome Is Highly Conserved across Geographic Locations and Distinct Chemical and Microbial Environments in New Zealand's Taupo Volcanic Zone.

UNLABELLED: Chthonomonas calidirosea T49(T) is a low-abundance, carbohydrate-scavenging, and thermophilic soil bacterium with a seemingly disorganized genome. We hypothesized that the C. calidirosea genome would be highly responsive to local selection pressure, resulting in the divergence of its genomic content, genome organization, and carbohydrate utilization phenotype across environments. We tested this hypothesis by sequencing the genomes of four C. calidirosea isolates obtained from four separate geothermal fields in the Taupo Volcanic Zone, New Zealand. For each isolation site, we measured physicochemical attributes and defined the associated microbial community by 16S rRNA gene sequencing. Despite their ecological and geographical isolation, the genome sequences showed low divergence (maximum, 1.17%). Isolate-specific variations included single-nucleotide polymorphisms (SNPs), restriction-modification systems, and mobile elements but few major deletions and no major rearrangements. The 50-fold variation in C. calidirosea relative abundance among the four sites correlated with site environmental characteristics but not with differences in genomic content. Conversely, the carbohydrate utilization profiles of the C. calidirosea isolates corresponded to the inferred isolate phylogenies, which only partially paralleled the geographical relationships among the sample sites. Genomic sequence conservation does not entirely parallel geographic distance, suggesting that stochastic dispersal and localized extinction, which allow for rapid population homogenization with little restriction by geographical barriers, are possible mechanisms of C. calidirosea distribution. This dispersal and extinction mechanism is likely not limited to C. calidirosea but may shape the populations and genomes of many other low-abundance free-living taxa. IMPORTANCE: This study compares the genomic sequence variations and metabolisms of four strains of Chthonomonas calidirosea, a rare thermophilic bacterium from the phylum Armatimonadetes It additionally compares the microbial communities and chemistry of each of the geographically distinct sites from which the four C. calidirosea strains were isolated. C. calidirosea was previously reported to possess a highly disorganized genome, but it was unclear whether this reflected rapid evolution. Here, we show that each isolation site has a distinct chemistry and microbial community, but despite this, the C. calidirosea genome is highly conserved across all isolation sites. Furthermore, genomic sequence differences only partially paralleled geographic distance, suggesting that C. calidirosea genotypes are not primarily determined by adaptive evolution. Instead, the presence of C. calidirosea may be driven by stochastic dispersal and localized extinction. This ecological mechanism may apply to many other low-abundance taxa.

Thermorudis pharmacophila sp. nov., a novel member of the class Thermomicrobia isolated from geothermal soil, and emended descriptions of Thermomicrobium roseum, Thermomicrobium carboxidum, Thermorudis peleae and Sphaerobacter thermophilus.

An aerobic, thermophilic and cellulolytic bacterium, designated strain WKT50.2T, was isolated from geothermal soil at Waikite, New Zealand. Strain WKT50.2T grew at 53-76 °C and at pH 5.9-8.2. The DNA G+C content was 58.4 mol%. The major fatty acids were 12-methyl C18 : 0 and C18 : 0. Polar lipids were all linked to long-chain 1,2-diols, and comprised 2-acylalkyldiol-1-O-phosphoinositol (diolPI), 2-acylalkyldiol-1-O-phosphoacylmannoside (diolP-acylMan), 2-acylalkyldiol-1-O-phosphoinositol acylmannoside (diolPI-acylMan) and 2-acylalkyldiol-1-O-phosphoinositol mannoside (diolPI-Man). Strain WKT50.2T utilized a range of cellulosic substrates, alcohols and organic acids for growth, but was unable to utilize monosaccharides. Robust growth of WKT50.2T was observed on protein derivatives. WKT50.2T was sensitive to ampicillin, chloramphenicol, kanamycin, neomycin, polymyxin B, streptomycin and vancomycin. Metronidazole, lasalocid A and trimethoprim stimulated growth. Phylogenetic analysis of 16S rRNA gene sequences showed that WKT50.2T belonged to the class Thermomicrobia within the phylum Chloroflexi, and was most closely related to Thermorudis peleae KI4T (99.6% similarity). DNA-DNA hybridization between WKT50.2T and Thermorudis peleae DSM 27169T was 18.0%. Physiological and biochemical tests confirmed the phenotypic and genotypic differentiation of strain WKT50.2T from Thermorudis peleae KI4T and other members of the Thermomicrobia. On the basis of its phylogenetic position and phenotypic characteristics, we propose that strain WKT50.2T represents a novel species, for which the name Thermorudis pharmacophila sp. nov. is proposed, with the type strain WKT50.2T ( = DSM 26011T = ICMP 20042T). Emended descriptions of Thermomicrobium roseum, Thermomicrobium carboxidum, Thermorudis peleae and Sphaerobacter thermophilus are also proposed, and include the description of a novel respiratory quinone, MK-8 2,3-epoxide (23%), in Thermomicrobium roseum.

Role of cytokines and chemokines in itch.

Cytokines classically are secreted "messenger" proteins that modulate cellular function of immune cells. Chemokines attract immune cells to the site where they exert various functions in inflammation, autoimmunity or cancer. Increasing evidence is emerging that cytokines or chemokines can act as "neuro-modulators" by activating high-affinity receptors on peripheral or central neurons, microglia cells or Schwann cells. Very recently, cytokines have been shown to act as pruritogens in rodents and humans, while a role of chemokines in itch has thus far been only demonstrated in mice. Upon stimulation, cytokines are released by skin or immune cells and form a "bridge of communication" between the immune and nervous system. For some cytokines such as IL-31 and TSLP, the evidence for this role is strong in rodents. For cytokines such as IL-4, there is some convincing evidence, while for cytokines such as oncostatin M, IL-2, IL-6, IL-8 and IL-13, direct evidence is currently limited. Current clinical trials support the idea that cytokines and chemokines and their receptors or signalling pathways are promising targets for the future therapy of certain subtypes of itch.

Effect of long-term starvation on the survival, recovery, and carbon utilization profiles of a bovine Escherichia coli O157:H7 isolate from New Zealand.

The ability to maintain a dual lifestyle of colonizing the ruminant gut and surviving in nonhost environments once shed is key to the success of Escherichia coli O157:H7 as a zoonotic pathogen. Both physical and biological conditions encountered by the bacteria are likely to change during the transition between host and nonhost environments. In this study, carbon starvation at suboptimal temperatures in nonhost environments was simulated by starving a New Zealand bovine E. coli O157:H7 isolate in phosphate-buffered saline at 4 and 15°C for 84 days. Recovery of starved cells on media with different nutrient availabilities was monitored under aerobic and anaerobic conditions. We found that the New Zealand bovine E. coli O157:H7 isolate was able to maintain membrane integrity and viability over 84 days and that the level of recovery depended on the nutrient level of the recovery medium as well as the starvation temperature. In addition, a significant difference in carbon utilization was observed between starved and nonstarved cells.

When the Penny Drops: Understanding how social class influences speciality careers in the UK medical profession.

In the UK, the medical profession is socially exclusive and socially stratified as doctors from more advantaged backgrounds are more likely to train for specialities with more competitive entry. However, in research to date the causes and consequences of social stratification have been overlooked. We explore this subject here, drawing on a qualitative study comprising in-depth interviews with 30 medical students and doctors from less advantaged socio-economic backgrounds negotiating medical school and early careers. Using Bourdieu's 'theory of practice' we show how socialisation in the family and at school influences how aspirant medics from less advantaged backgrounds view the world, suggesting some inclination towards more community orientated careers, which may be less competitive. However, these tendencies are encouraged as they lack stocks of social, economic and cultural capital, which are convertible to power and position in the field. While allowing for both choice and constraint our core argument is that speciality outcomes are sometimes inequitable and potentially inefficient, as doctors from more advantaged backgrounds have privileged access to more competitive careers for reasons not solely related to ability or skill. Our main theoretical contribution is to literature in the sociology of medical education where ours is the first study to open-up the 'black box' of causal factors connecting medical students' resources on entering the field of education and training with speciality outcomes, though our findings also have important implications for practitioners, the profession and for patients. We discuss the implications for safe and effective healthcare and how this informs directions for future research.

Diffuse Lichen Planopilaris Masquerading as Diffuse Alopecia Areata.

Introduction: Lichen planopilaris (LPP) is a primary lymphocytic cicatricial alopecia that represents a form of follicular lichen planus. Case Presentation: We describe a case of coexisting diffuse LPP and female pattern hair loss masquerading as diffuse alopecia areata in a 32-year-old female. Discussion: In complex cases such as this, dermoscopy-guided vertical and horizontal biopsies from androgen sensitive and insensitive areas are helpful in increasing diagnostic yield. Prompt initiation of treatment is key to halting disease progression. Long-term follow-up is important as resolution of clinical signs does not always correlate with the absence of disease progression.

Investigating the lignocellulolytic gut microbiome of huhu grubs (Prionoplus reticularis) using defined diets and dietary switch.

The huhu beetle (Prionoplus reticularis) is the largest endemic beetle found throughout Aotearoa New Zealand, and is characterised by feeding on wood during its larval stage. It has been hypothesised that its gut microbiome plays a fundamental role in the degradation of wood. To explore this idea we examined the fungal and bacterial community composition of huhu grubs' frass, using amplicon sequencing. Grubs were reared on an exclusive diet of either a predominantly cellulose source (cotton) or lignocellulose source (pine) for 4 months; subsequently a diet switch was performed and the grubs were grown for another 4 months. The fungal community of cellulose-reared huhu grubs was abundant in potential cellulose degraders, contrasting with the community of lignocellulose-reared grubs, which showed abundant potential soft rot fungi, yeasts, and hemicellulose and cellulose degraders. Cellulose-reared grubs showed a less diverse fungal community, however, diet switch from cellulose to lignocellulose resulted in a change in community composition that showed grubs were still capable of utilising this substrate. Conversely, diet seemed to have a limited influence on huhu grub gut bacterial communities.

A genus in the bacterial phylum Aquificota appears to be endemic to Aotearoa-New Zealand.

Allopatric speciation has been difficult to examine among microorganisms, with prior reports of endemism restricted to sub-genus level taxa. Previous microbial community analysis via 16S rRNA gene sequencing of 925 geothermal springs from the Taupo Volcanic Zone (TVZ), Aotearoa-New Zealand, revealed widespread distribution and abundance of a single bacterial genus across 686 of these ecosystems (pH 1.2-9.6 and 17.4-99.8 °C). Here, we present evidence to suggest that this genus, Venenivibrio (phylum Aquificota), is endemic to Aotearoa-New Zealand. A specific environmental niche that increases habitat isolation was identified, with maximal read abundance of Venenivibrio occurring at pH 4-6, 50-70 °C, and low oxidation-reduction potentials. This was further highlighted by genomic and culture-based analyses of the only characterised species for the genus, Venenivibrio stagnispumantis CP.B2T, which confirmed a chemolithoautotrophic metabolism dependent on hydrogen oxidation. While similarity between Venenivibrio populations illustrated that dispersal is not limited across the TVZ, extensive amplicon, metagenomic, and phylogenomic analyses of global microbial communities from DNA sequence databases indicates Venenivibrio is geographically restricted to the Aotearoa-New Zealand archipelago. We conclude that geographic isolation, complemented by physicochemical constraints, has resulted in the establishment of an endemic bacterial genus.

PXS-4681A, a potent and selective mechanism-based inhibitor of SSAO/VAP-1 with anti-inflammatory effects in vivo.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Diversity of Methylobacterium species associated with New Zealand native plants.

Methylobacterium species are abundant colonizers of the phyllosphere due to the availability of methanol, a waste product of pectin metabolism during plant cell division. The phyllosphere is an extreme environment, with a landscape that is heterogeneous and continuously changing as the plant grows and is exposed to high levels of ultraviolet irradiation. Geographically, New Zealand (NZ) has been isolated for over a million years, has a biologically diverse flora, and is considered a biodiversity hotspot, with most native plants being endemic. We therefore hypothesize that the phyllosphere of NZ native plants harbor diverse groups of Methylobacterium species. Leaf imprinting using methanol-supplemented agar medium was used to isolate bacteria, and diversity was determined using ARDRA and 16S rRNA gene sequencing. Methylobacterium species were successfully isolated from the phyllosphere of 18 of the 20 native NZ plant species in this study, and six different species were identified: M. marchantiae, M. mesophilicum, M. adhaesivum, M. komagatae, M. extorquens, and M. phyllosphaerae. Other α, ß, and γ-Proteobacteria, Actinomycetes, Bacteroidetes, and Firmicutes were also isolated, highlighting the presence of other potentially novel methanol utilizers within this ecosystem. This study identified that Methylobacterium are abundant members of the NZ phyllosphere, with species diversity and composition dependent on plant species.