The hereditary transmission of the glutathione transferase hGSTT1-1 conjugator phenotype in a large family

The polymorphism of human glutathione transferase hGSTT1-1 is expressed in three phenotypes. Experimentally, individuals can be classified as non-conjugators, low conjugators and 'high' conjugators depending on the enzyme activity in blood towards methylene chloride using a gas chromatographic assay. Non-conjugators do not have a functional hGSTT1 gene; however, little is known about the molecular basis of the three conjugator phenotypes. The higher hGSTT1-1 activity in high conjugators may be the result of enzyme induction or be genetically determined. Twenty-nine members of a large family, including three generations were phenotyped and genotyped with respect to hGSTT1-1. The hGSTT1-1 enzyme activity of high conjugators was twice as high as that of low conjugators. The distribution of hGSTT1-1 phenotypes strongly indicates a Mendelian intermediary inheritance, in which a gene-dosage effect results in a doubled enzyme expression in the presence of two functional alleles. The Mendelian intermediary inheritance is further supported by the findings of a semiquantitative polymerase chain reaction method designed to distinguish the three genotypes of hGSTT1 for rapid screening of large study groups.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing,CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, preexisting SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SClike BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (ARlow) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The ARlow seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

B-lactamase-mediated resistance to antimicrobials : the relationship between genotype and phenotype

This thesis examined the ability to predict the emergence of bacteria resistant to antibiotics using genetic markers in the bacteria. Bacteria containing the genetic markers were able to become resistant to antibiotics, whereas bacteria that did not have the genetic markers remained susceptible. Existing techniques can identify the presence of resistance by looking at the characteristics of the bacteria during growth. However, having the ability to predict antibiotic resistance before it emerges could improve the preservation of currently available antibiotics and minimise treatment failure.

Coordination and plasticity in leaf anatomical traits of invasive and native vine species

Premise of the study: Plant invasiveness can be promoted by higher values of adaptive traits (e.g., photosynthetic capacity, biomass accumulation), greater plasticity and coordination of these traits, and by higher and positive relative influence of these functionalities on fitness, such as increasing reproductive output. However, the dataset for this premise rarely include linkages between epidermal-stomatal traits, leaf internal anatomy, and physiological performance. Methods: Three ecological pairs of invasive vs non-invasive (native) woody vine species of South-East Queensland, Australia were investigated for trait differences in leaf morphology and anatomy under varying light intensity. The linkages of these traits with physiological performance (e.g. water use efficiency, photosynthesis, and leaf construction cost) and plant adaptive traits of specific leaf area, biomass, and relative growth rates were also explored. Key results: Mean leaf anatomical trait differed significantly between the two groups, except for stomatal size. Plasticity of traits, and to a very limited extent, their phenotypic integration were higher in the invasive relative to the native species. ANOVA, ordination, and analysis of similarity suggest that for leaf morphology and anatomy, the three functional strategies contribute to the differences between the two groups in the order phenotypic plasticity > trait means > phenotypic integration. Conclusions: The linkages demonstrated in the study between stomatal complex/gross anatomy and physiology are scarce in the ecological literature of plant invasiveness, but the findings suggest that leaf anatomical traits need to be considered routinely as part of weed species assessment and in the worldwide leaf economic spectrum.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

A high and low fear phenotype mouse model

Fear-related illnesses such as post-traumatic stress disorder (PTSD) impose a tremendous burden on individual quality of life, families, and the national economy. In the military population, 17-20% of services members returning from deployment are diagnosed with PTSD. While treatments have improved for PTSD and are helpful for some, many people continue to suffer despite therapy. The aim of this research is to examine fear memory behaviourally and at the cellular level in the amygdala by using a unique inter-cross strain of high and low fear phenotype mice. An extended outcross C57BL/6J x DBA/2J (F8) are selected for high or low Pavlovian fear memory to context and cue. On presentation of either the original learning context or the cue (tone) mice display high or low levels of freezing as a behavioural measure of fear. In order to identify key aspects of the cellular basis of this difference in fear memory behaviour we are making measurements of protein levels and neuron numbers of a known pathway involved in the consolidation of a long term fear memory (pMAPK). Ongoing studies aim to determine if high fear behaviour is associated with differential signalling in the lateral amygdala compared to low fear behaviour. Additionally, by blocking this pathway in the lateral amygdala (LA), we aim to reduce fear behaviour following Pavlovian fear conditioning. This research will help to unravel the cellular mechanisms underlying high fear behaviour and advance the field toward targeted treatment and improved outcomes, ultimately improving human quality of life.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Androgen-targeted therapy-induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer : an opportunity for intervention

Androgens regulate biological pathways to promote proliferation, differentiation, and survival of benign and malignant prostate tissue. Androgen receptor (AR) targeted therapies exploit this dependence and are used in advanced prostate cancer to control disease progression. Contemporary treatment regimens involve sequential use of inhibitors of androgen synthesis or AR function. Although targeting the androgen axis has clear therapeutic benefit, its effectiveness is temporary, as prostate tumor cells adapt to survive and grow. The removal of androgens (androgen deprivation) has been shown to activate both epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD) programs. EMT has established roles in promoting biological phenotypes associated with tumor progression (migration/invasion, tumor cell survival, cancer stem cell-like properties, resistance to radiation and chemotherapy) in multiple human cancer types. NEtD in prostate cancer is associated with resistance to therapy, visceral metastasis, and aggressive disease. Thus, activation of these programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression. Brachyury, Axl, MEK, and Aurora kinase A are molecular drivers of these programs, and inhibitors are currently in clinical trials to determine therapeutic applications. Understanding tumor cell plasticity will be important in further defining the rational use of androgen-targeted therapies clinically and provides an opportunity for intervention to prolong survival of men with metastatic prostate cancer.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF‑2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF‑2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β‑glycerophosphate). FGF‑2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type Ⅰ, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF‑2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT‑qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF‑2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF‑2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

High and low fear phenotypes show differences in phosphorylated (p44/42 ERK) MAPK-expressing neurons in the lateral amygdala

Post traumatic stress disorder (PTSD) is a serious medical condition effecting both military and civilian populations. While its etiology remains poorly understood it is characterized by high and prolonged levels of fear responding. One biological unknown is whether individuals expressing high or low conditioned fear memory encode the memory differently and if that difference underlies fear response. In this study we examined cellular mechanisms that underlie high and low conditioned fear behavior by using an advanced intercrossed mouse line (B6D2F1) selected for high and low Pavlovian fear response. A known requirement for consolidation of fear memory, phosphorylated mitogen activated protein kinase (p44/42 (ERK) MAPK (pMAPK)) in the lateral amygdala (LA) is a reliable marker of fear learning-related plasticity. In this study, we asked whether high and low conditioned fear behavior is associated with differential pMAPK expression in the LA and if so, is it due to an increase in neurons expressing pMAPK or increased pMAPK per neuron. To examine this, we quantified pMAPK-expressing neurons in the LA at baseline and following Pavlovian fear conditioning. Results indicate that high fear phenotype mice have more pMAPK-expressing neurons in the LA. This finding suggests that increased endogenous plasticity in the LA may be a component of higher conditioned fear responses and begins to explain at the cellular level how different fear responders encode fear memories. Understanding how high and low fear responders encode fear memory will help identify novel ways in which fear-related illness risk can be better predicted and treated.

Variable phenotype in 16p duplication within a family

Background More individuals are now being identified with very rare genetic syndromes. We present a family with an inherited duplication of 16p11.2 to 16q12.1 in ring formation. Three of the four children, (aged 15 months to 10 years), mother, uncle, and grandmother are affected. Our aim was to provide preliminary evidence of possible phenotypic patterns of learning and behaviour associated with this chromosome anomaly. Method Psychometric assessments were undertaken with all four children. The mother and uncle also agreed to participate in the study. Measures of development (Bayley or Mullen), intellectual ability (WISC-IV or WAIS-III), academic achievement (WIAT-II), adaptive behaviour (Vinelands), and other relevant aspects of functioning (e.g., Children’s Memory Scale) were administered. Results. The first-born child is the only one who is unaffected. Her intellectual ability was assessed as being within the superior range. The second child experienced early difficulties with speech and motor skills. Although his intelligence is average, he has learning difficulties and significant auditory memory problems. The third child’s speech and motor milestones were markedly delayed. He has a complex medical history that includes a vitamin B12 deficiency. On the Mullen Scales at age 4 his scores ranged from average to very low. The development of the youngest child (aged 15 months), who also had a B12 deficiency but was treated early, was assessed as being within typical limits. Conclusions There is considerable developmental variability among the three children with this inherited 16p duplication. We discuss the intriguing similarities and differences, considering common features that may reflect phenotypic patterns and speculating about possible explanations for the variable presentations.

Identification of genetic variants contributing to the migraine phenotype in different Australian populations

This project aimed to identify novel genetic risk variants associated with migraine in the Norfolk Island population. Statistical analysis and bioinformatics approaches such as polygenic modeling and gene clustering methods were carried out to explore genotypic and expression data from high-throughput techniques. This project had a particular focus on hormonal genes and other genetic variants and identified a modest effect size on the migraine phenotype.

IgE+ B cells are scarce, but allergen-specific B cells with a memory phenotype circulate in patients with allergic rhinitis

Background Despite the critical role of immunoglobulin E (IgE) in allergy, circulating IgE+ B cells are scarce. Here, we describe in patients with allergic rhinitis B cells with a memory phenotype responding to a prototypic aeroallergen. Methods Fifteen allergic rhinitis patients with grass pollen allergy and 13 control subjects were examined. Blood mononuclear cells stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) were cultured with Bahia grass pollen. Proliferation and phenotype were assessed by multicolour flow cytometry. Results In blood of allergic rhinitis patients with high serum IgE to grass pollen, most IgEhi cells were CD123+ HLA-DR- basophils, with IgE for the major pollen allergen (Pas n 1). Both B and T cells from pollen-allergic donors showed higher proliferation to grass pollen than nonallergic donors (P = 0.002, and 0.010, respectively), whereas responses to vaccine antigens and mitogen did not differ between groups. Allergen-driven B cells that divided rapidly (CD19mid CD3- CFSElo) showed higher CD27 (P = 0.008) and lower CD19 (P = 0.004) and CD20 (P = 0.004) expression than B cells that were slow to respond to allergen (CD19hi CD3- CFSEmid). Moreover, rapidly dividing allergen-driven B cells (CD19mid CFSElo CD27hi) showed higher expression of the plasmablast marker CD38 compared with B cells (CD19hi CFSEmid CD27lo) that were slow to divide. Conclusion Patients with pollen allergy but not control donors have a population of circulating allergen-specific B cells with the phenotype and functional properties of adaptive memory B-cell responses. These cells could provide precursors for allergen-specific IgE production upon allergen re-exposure. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.