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Purpose Hormonal and inflammatory factors have been suggested as potentially influencing depressive state and depressive symptoms, but rarely compared for their relative contribution to these states and to specific depressive symptoms. This study examined cortisol:C-reactive protein (CRP) ratio, plus cortisol and CRP separately, as correlates of global depression and fatigue-related depression. Patients and methods One hundred and twenty-six community volunteers from rural Australia provided saliva and serum samples, and also completed a depression inventory. Results There was a significant correlation between cortisol:CRP ratio and depression-related fatigue, and this resolved to the effects of CRP rather than cortisol. Most of the variance in this association came from patients who were "depressed", and there were no significant gender associations. Conclusion Inflammation, rather than HPA-axis activity, was associated with depression-related fatigue, supporting a model that places inflammation as a contributor to one of the major symptoms and predictors of depression. Individualization of therapy for depression-related fatigue in chronically stressed or physically ill patients might benefit from future research into cytokine therapy.

Endosperm of rice and other cereals accumulates high concentrations of the predominant in planta auxin, indole-3-acetic acid (IAA) during early grain development. However, IAA signalling and function during endosperm development are poorly understood. Here, we report that OsYUC12 (an auxin biosynthesis gene) and OsIAA29 (encoding a non-canonical AUX/IAA) are both expressed exclusively in grains, reaching a maximum 5 to 6 days after pollination. OsYUC12 expression is localized in the aleurone, sub-aleurone and embryo, whereas OsIAA29 expression is restricted to a narrow strip in the dorsal aleurone, directly under the vascular bundle. Although rice has been reported to lack endosperm transfer cells (ETCs), this region of the aleurone is enriched with sugar transporters and is likely to play a key role in apoplastic nutrient transfer, analogous to ETCs in other cereals. OsIAA29 has orthologues only in grass species; expression of which is also specific to early grain development. OsYUC12 and OsIAA29 are temporally co-expressed with two genes (AL1 and OsPR602) previously linked to the development of dorsal aleurone or ETCs. Also up regulated at the same time are a cluster of MYB-related genes (designated OsMRPLs) homologous to ZmMRP-1, which regulates maize ETC development. Wheat homologues of ZmMRP-1 are also expressed in ETCs. Although previous work has suggested that other cereals do not have orthologues of ZmMRP-1, our work suggests OsIAA29 and OsMRPLs and their homologues in other grasses are part of an auxin-regulated, conserved signalling network involved in the differentiation of cells with ETC-like function in developing cereal grains.Main ConclusionNon-canonical AUX/IAA protein, OsIAA29, and ZmMPR-1 homologues, OsMRPLs, are part of an auxin-related signalling cascade operating in the dorsal aleurone during early rice grain development.

Christopher F Sharpley,1 Vicki Bitsika,2 Mary E McMillan,1 Emmanuel Jesulola,1 Linda L Agnew1 1Brain-Behaviour Research Group, University of New England, Armidale, NSW, Australia; 2Centre for Autism Spectrum Disorders, Bond University, Gold Coast, QLD, Australia Purpose: Depression remains difficult to treat in all cases, and further investigation of the role of genetic and environmental factors may be valuable. This study was designed to investigate the association between the short (s) versus non-short (non-s) 5HTTLPR variants, presence of childhood stressors and recent life stressors, and depression, and to do so at two levels that would expose the associations between total depression scores and also individual depression items.Materials and methods: Two hundred and forty-nine volunteers from one of the Australian Electoral Office electorates covering a large rural land area completed a series of questionnaires about childhood and recent life stress and depression, and provided a buccal cell sample for genotyping the 5-HTTLPR polymorphism into s versus non-s carriers.Results: Although there were no significant differences in the depression scores of the s-carriers versus the non-s carriers, each subtype of the 5-HTTLPR polymorphism showed different patterns of association between childhood stress and depression symptoms, and between recent life stress and depression symptoms.Conclusion: Individualization of therapy for depression may be achieved through consideration of the specific associations that patients exhibit between life stress, 5-HTTLPR polymorphism, and depression symptomatology. Keywords: genes, mental health, life experiences

SUMMARY A series of studies were undertaken to optimize the propagation of hemorrhagic enteritis virus (HEV) in specific-pathogen-free (SPF) chickens. A total of 562 SPF chickens were orally inoculated with an Australian avirulent HEV isolate of turkey origin at 9, 14, 21, or 28 days of age with 5, 6, 7, or 8 log 10 genomic copies (GC), while 102 chickens served as uninfected controls. No clinical signs were observed in infected chickens. There was an inoculum-dose–dependent increase in the relative spleen and liver weight (P < 0.01). Relative spleen weight 7 days post-HEV inoculation was up to 85% higher in chickens that were inoculated with 6 to 7 GC compared with controls, with no further increase at higher doses. Relative liver weight increased up to 14% in chickens inoculated with 6 GC, with no further increase. Birds inoculated with a 7 GC dose had a higher frequency of HEV DNA-positive birds (77% to 86%) than birds inoculated with lower doses (33% to 59%; P < 0.01). The most efficient dose for live...

Small molecular weight oxygen free radical species (ROS) involved in oxidative stress can cause damage to cellular macromolecules including proteins, DNA and lipids. One of the most important enzymes involved in ROS detoxification is glutathione peroxidase (GPx). Here we study the age-related expression of GPx isoenzymes in various parts of the rat kidney with and without exposure to external oxidative stress. These results are correlated to the age dependent changes in the expression of the chaperone, Hsp-70. Protein and mRNA expression of GPx1 and GPx4 was studied in different regions of the kidney in ageing rats in the presence and absence of the external stressor 0.2 mM H2O2. Protein levels were examined by Western blot analysis following detection with appropriate antibodies and mRNA levels were analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using appropriate primer sequences. mRNA expression for the chaperone Hsp70 was investigated in parallel. After reaching a peak at maturity (12 weeks), GPx1 protein and mRNA levels decreased with age under both control and stress conditions, and were higher in the cortex than in the outer and inner medulla. GPx4 protein and mRNA levels showed few comparable age-related changes. By contrast with the observed age-related decrease in GPx1 expression, chaperone Hsp-70 mRNA expression greatly increased with age. These findings suggest that the age-related decline in GPx1 expression in the cortex may be partly offset by a reciprocal change in Hsp-70 expression. These results are consistent with the oxidative stress theory of ageing.

Haemorrhagic enteritis virus (HEV) causes clinical haemorrhagic enteritis in young poults and/or subclinical immunosuppression which is often associated with colibacillosis. This disease is controlled with live vaccines worldwide, however, importation of HEV vaccines or cells that support HEV propagation are not permitted in Australia. A major experiment in isolators was conducted to test the safety and efficacy of a putative HEV vaccine. The study had a factorial design with four factors namely vaccination age (28 and 42 days of age), vaccine dose (0, 105 , 106 , 107 genomic copies of HEV vaccine), challenge with HEV (yes, no) and vaccination-challenge interval (7, 21 or 42 days). A total of 315 poults were used providing 6-8 birds per treatment combination. Turkey growth rate, mortality, pathological findings, anti-HEV antibodies and viral load were examined. Vaccination lead to significant increases in anti HEV antibody over the following 2-4 weeks. Overall, vaccination with 106 and 107 was protective against increase in relative splenic weight and splenic viral load in challenged birds. Clinical haemorrhagic enteritis was not induced by any treatment but there was an increased incidence of airsacculitis in groups receiving either HEV vaccine or challenge virus compared to the negative control birds (25.8-29.3% vs. 9.4%, P

This study aimed to investigate the feasibility of propagating and titrating hemorrhagic enteritis virus (HEV) in chicken embryos. A total of 308 embryonated eggs were used. At 10 days of embryonic age, eggs were inoculated via allantoic sac or chorioallantoic membrane routes with non-heat-treated (live) HEV or heat-treated (dead) HEV or served as negative controls. Allantoic fluid retrieved at 0, 1, 3, 5, and 7 days postinoculation (dpi) was tested for HEV by quantitative PCR. Inoculation with HEV did not cause visible growth impairment or lesions in the chicken embryos. Overall, there was no difference in postinoculation mortality rates among groups sham-inoculated (6/30, 20.0%) or inoculated with live (34/252, 13.4%) or dead (3/ 26, 6.9%) HEV (El virus de la enteritis hemorrágica de los pavos puede ser titulado pero no propagado en embriones de pollo. Este estudio tuvo como objetivo investigar la viabilidad de propagar y titular al virus de la enteritis hemorrágica de los pavos (con las siglas en inglés HEV) en embriones de pollo. Se utilizaron un total de 308 huevos embrionados. A los 10 días de edad embrionaria, los huevos se inocularon por vía saco alantoideo o por la membrana corioalantoidea con el virus de la enteritis hemorrágica sin tratamiento térmico (vivo) o con un virus de la enteritis hemorrágica con tratamiento térmico (muerto) y algunos sirvieron como controles negativos. El fluido alantoideo recuperado a los cero, uno, tres, cinco y siete días después de la inoculación se analizó para detectar el VHE mediante un método cuantitativo de PCR. La inoculación con el virus de la enteritis hemorrágica no causó daños visibles en el crecimiento ni lesiones en los embriones de pollo. En general, no hubo diferencias en las tasas de mortalidad después de la inoculación entre los grupos con inoculación simulada (6/30, 20.0%) o inoculados con el virus vivo de la enteritis hemorrágica (34/252, 13.4%) o con el virus muerto (3/26, 6.9%) (

Molecular methods have been used in the study of Azospirillum and other related PGPRs to carry out gene functional analysis, create gene knockouts, generate genetically engineered strains, and carry out gene expression studies. Genetic transformation has routinely been carried out using conjugation, while chromosomal modifications have been performed using unstable, suicide plasmids, or more stable, broad host-range vectors. Gene expression studies are often carried out using promoter-bound reporter genes; however, quantitative methods such as reverse transcribed polymerase chain reaction can now be used to directly study gene expression. In this chapter we describe the common types of vectors used in Azospirillum, as well as methods for transformation and mutagenesis. We also describe the use of promoter-bound reporter genes and the applications of quantitative RT-PCR for Azospirillum gene expression studies. Methods for the isolation of DNA and RNA from Azospirillum for use in molecular and gene expression studies are also described.

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Spermatogonial stem cells hold enormous potential in mammalian reproductive medicine through the preservation of gametes, the restoration of fertility, enhancement of germ-lineage genetic manipulation and the improvement in our understanding of stem cell biology. Here we describe the protein profiles of the membrane compartment of bovine testicular cell isolates which were enriched for germ cells using differential plating. The isolated cells were characterised with antibodies to UCHL1 (previously known as PGP9.5) for type A spermatogonia; DDX4 (previously known as VASA) for germ cells and vimentin for Sertoli cells. Ultracentrifugation techniques were used to specifically isolate cell membranes, with membrane protein identifications significantly increased when compared to whole cell lysates. We utilised the filter-aided sample preparation protocol for improved digestion efficiency of membrane proteins. Using ESI-LC-MS/MS, we compared the proteins present in two cell populations. A total of 1,387 proteins were identified in bovine testis cell isolates, of which 39% were membrane-associated. A total of 64 proteins were differentially expressed in the non-adhered fraction (enriched for undifferentiated germ cells), of which 16 were unique to this cell population and the remaining 48 showed a two-fold change as judged by spectral counting. This analysis revealed a number of candidate germ cell markers including the known markers, DDX4 and UCHL1. The proteomic profiles generated in this study support and complement transcriptomics studies and reinforce the potential of proteomics in identifying and characterising the protein effectors of self-renewal and/or differentiation in stem cells. Normal 0 false false false EN-AU X-NONE X-NONE

Adhesion molecules are expressed by both adult and embryonic stem cells, with different classes of adhesion molecules involved in cell-membrane and intercellular contacts. In this study the expression of the adhesion molecule claudin-8 (CLDN8), a tight-junction protein, was investigated as a potential marker for undifferentiated spermatogonia in the bovine testis. We found that CLDN8 was expressed by both spermatogonia and a subset of Sertoli cells in the bovine testis. We also showed co-expression of GFRα1 in testis cells with CLDN8 and with Dolichos biflorus agglutinin–fluorescein isothiocyanate (DBA–FITC) staining. We observed co-enrichment of spermatogonia and CLDN8-expressing Sertoli cells in DBA–FITC-assisted magnetic-activated cell sorting (MACS), an observation supported by results from fluorescence-activated cell sorting analysis, which showed CLDN8-expressing cells were over-represented in the MACS-positive cell fraction, leading to the hypothesis that CLDN8 may play a role in the spermatogonial stem-cell niche.

Nonviral reprogramming of bovine embryonic and adult fibroblasts was undertaken using electroporation of a polycistronic plasmid carrying human reprogramming factors Oct4, Sox2, Lin28, and Nanog (Addgene Plasmid #20922). Because of difficulties encountered in reliably and reproducibly reprogramming bovine cells to a pluripotent state, several small-molecule combinations have been tested to support reprogramming. Previously, a combination of three molecules, (sodium butyrate, PD0325901 and SB431542; NaB-PD-SB) in induced pluripotent stem (iPS) medium [Minimum Essential Medium Alpha, 20% FBS, 1x insulin-transferrin-selenium (ITS), 2 mM Glutamax (Gibco, Grand Island, NY, USA), 100 μM nonessential amino acids (NEAA), 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukaemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the reprogramming process of bovine iPS cells, with colonies observed at 12 days post-electroporation, as opposed to 21 days without the addition of the small-molecule cocktail. The addition of 1 mM valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, in combination with the other small-molecule cocktail (NaB-PD-SB), was found to improve the reprogramming efficiency of bovine adult and embryonic fibroblasts, with colonies observed at Day 10 post-electroporation, and the average number of colonies present per 10-cm dish, rising from an average of 5 colonies observed in the NaB-PD-SB cocktail to 27 with the addition of VPA. Colonies grown in the presence of VPA had a consistent morphology, forming compact domed colonies consisting of small round cells with well-defined borders. It was observed that colonies growing in the presence of VPA tended to have a better defined border and grew larger in size than those grown in the presence of NaB-PD-SB alone. Colonies began to differentiate after 21 days and were no longer alkaline phosphatase positive after this time. Cells were either harvested for mRNA extraction or differentiated into embryoid bodies (EB). Expression of pluripotency genes, Oct4, Sox2, Klf4, Nanog, and Alkaline Phosphatase were significantly increased in the presence of VPA compared to nonreprogrammed somatic bovine fibroblasts, with expression profiles similar to those grown in the NaB-PD-SB cocktail. Embryoid bodies were analysed for gene expression of different germ layer markers, FOXA2 (endoderm), Nestin and TUBB3 (ectoderm), and Desmin (mesoderm) using quantitative RT–PCR. For both EBs derived from the NaB-PD-SB cocktail and those with VPA, at least one marker from each germ layer was present, demonstrating the potential potency of these cells. At least a 10-fold increase in expression of these was observed in comparison to somatic fibroblast cells. It is apparent that the addition of small molecules can assist in the reprogramming of bovine iPS cells, and addition of VPA to the cocktail results in more consistent putative bovine iPS colonies. Further work is needed to identify the causes for early differentiation of colonies in order to obtain fully reprogrammed pluripotent stem cells.

Electron-microscopic examination of an oat-cell carcinoma associated with the ectopic ACTH syndrome demonstrated characteristic cytoplasmic granules of 60-240 nm. diameter, consisting of a dense central core separated by a clear halo from an outer investing membrane. Comparison with previously examined non-secretory oat-cell carcinomas showed the granules to be more numerous in the present case. They are considered to represent secretory activity in the tumour. ImagesFigs. 1-2

Secretion rates of dehydroepiandrosterone and its sulphate were measured in three cases of ‘testicular feminization”. The pattern of secretion rates was found to be male, rather than female. Plasma levels of 17-hydroxycorticoids (17-OHCS) and 17-oxosteroids (17-KS) were determined in five patients. The 17-KS were found to be at the upper limit of the normal female range and overlapping the male range. 17-OHCS were found to be at the lower end of the female range. Plasma testosterone levels were definitely in the male range. Total urinary oestrogens were at the upper limit of the male range and lower than the normal female range. However, they were higher than those found in women with primary amenorrhoea. The administration of both ACTH and HCG resulted in a rise in the titre of total urinary oestrogens; however, the rise after HCG administration was greater than that after ACTH treatment. The failure to explain the female phenotype in terms of plasma and urinary hormone studies is discussed.

There is a growing body of evidence that demonstrates the potential of various microbes to enhance plant productivity and yield in cropping systems. Realizing the potential of beneficial microbes requires an understanding of the role of microbes in growth promotion, particularly in terms of fertilization and disease control, the underlying mechanisms and the challenges in application and commercialization of plant growth-promoting (PGP) microbes. This review focuses specifically on the use of PGP microbes in the cotton industry and summarizes the commercial bioinoculant products currently available for cotton; highlighting factors that must be considered for future development of PGP microbial products for the cotton industry. Given the paucity of information on beneficial microbes for cotton production systems in comparison to those for other cropping systems (e.g. legumes and grains), a snapshot of the current research is critical in light of the increased interest in cotton inoculants, mainly in developing countries such as India, and the overall increased interest in PGP applications as part of promoting sustainable agriculture.