Visualising single molecules of HIV-1 and miRNA nucleic acids

Background
The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length.

Results
We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.

Conclusions
Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Maternal dietary supplementation with saturated, but not monounsaturated or polyunsaturated fatty acids, leads to tissue-specific inhibition of offspring Na+,K+-ATPase

In rats, a maternal diet rich in lard is associated with reduced Na+,K+-ATPase activity in adult offspring kidney. We have addressed the role of different fatty acids by evaluating Na+,K+-ATPase activity in offspring of dams fed diets rich in saturated (SFA), monounsaturated (MUFA) or polyunsaturated (PUFA) fatty acids. Female Sprague–Dawley rats were fed, during pregnancy and suckling, a control diet (4% w/w corn oil) or a fatty acid supplemented diet (24% w/w). Offspring were reared on chow (4% PUFA) and studied at 6 months. mRNA expression (real-time PCR) of Na+,K+-ATPase α subunit and protein expression of Na+,K+-ATPase subunits (Western blot) were assessed in kidney and brain. Na+,K+-ATPase activity was reduced in kidney (P < 0.05 versus all groups) and brain (P < 0.05 versus control and MUFA offspring) of the SFA group. Neither Na+,K+-ATPase α1 subunit mRNA expression, nor protein expression of total α, α1, α2, α3 or β1 subunits were significantly altered in kidney in any dietary group. In brains of SFA offspring α1 mRNA expression (P < 0.05) was reduced compared with MUFA and PUFA offspring, but not controls. Also in brain, SFA offspring demonstrated reduced (P < 0.05) α1 subunit protein and increased phosphorylation (P < 0.05) of the Na+,K+-ATPase modulating protein phospholemman at serine residue 63 (S63 PLM). Na+,K+-ATPase activity was similar to controls in heart and liver. In utero and neonatal exposure to a maternal diet rich in saturated fatty acids is associated with altered activity and expression of Na+,K+-ATPase in adulthood, but mechanisms appear tissue specific.

Isolation and characterisation of omega-3 fatty acids producing marine microbes

As a renewable alternative to fish oil, microbial-derived omega-3-fatty acids can be potential nutritional supplements. This study reported the isolation of novel oleaginous marine microbes from the Victorian marine environment capable of producing omega-3-fatty acids and other bio-actives. Fermentation strategies using low cost substrates improved omega-3-oils production in selected isolates.

Relationship between membrane fatty acids and cognitive symptoms and information processing in individuals at ultra-high risk for psychosis

Cognitive symptoms and impairment are central to schizophrenia and often an early sign of this condition. The present study investigated biological correlates of cognitive symptoms and performance in individuals at ultra-high risk (UHR) for psychosis. The study sample comprised 80 neuroleptic-naïve UHR individuals aged 13-25 years. Associations among erythrocyte membrane fatty acid levels, measured by gas chromatography, and cognitive functioning were investigated in UHR patients. Subjects were divided into terciles based on their scores on the cognitive factor of the Positive and Negative Syndrome Scale. The Zahlen-Verbindungs Test (ZVT) (the number-combination test) was also used as a measure of information-processing speed. Exploratory analysis was conducted to investigate the relationship between membrane fatty acid levels with the size of the intracranial area (ICA), a neurodevelopmental measure relevant to schizophrenia, in half of subjects (n=40) using magnetic resonance imaging. The adjusted analysis revealed that omega-9 eicosenoic and erucic acid levels were significantly higher, but omega-3 docosahexaenoic acid levels were significantly lower, in the cognitively impaired than in the cognitively intact group. We found a significant negative association of eicosenoic, erucic, and gamma-linoleic acids with ZVT scores. A negative association between ICA and membrane levels of eicosenoic acid was also found. This is the first study to demonstrate the relationship between membrane fatty acids and cognitive function in neuroleptic-naïve subjects at UHR for psychosis. The study findings indicate that abnormalities in membrane fatty acids may be associated with the neurodevelopmental disruption associated with the cognitive impairments of individuals at UHR for psychosis.

Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner

Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p < 0.01, 30 μM) and 13 HODE (p < 0.01, 30 μM), and the equivalent cell viability was also decreased (p < 0.001). Both 9-HODE and 13-HODE (but not LA or ALA) markedly increased caspase-3/7 activity (p < 0.001) in both monocytes and adherent THP-1 cells, with 9-HODE the more potent. In addition, 9-HODE and 13-HODE both increased Annexin-V labelling of cells (p < 0.001). There was no effect of LA, ALA, or the PPARγ agonist rosiglitazone (1μM), but the effect of HODEs was replicated with apoptosis-inducer camptothecin (10μM). Only 9-HODE increased DNA fragmentation. The pro-apoptotic effect of HODEs was blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent—and specific—regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

Randomized controlled trial examining the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells

The present randomized, placebo-controlled, double-blind, parallel-groups clinical trial examined the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells. Healthy adult humans (n = 160) were randomized to receive 6 g of fish oil, 6 g of fish oil plus a multivitamin, 3 g of fish oil plus a multivitamin or a placebo daily for 16 weeks. Treatment with 6 g of fish oil, with or without a daily multivitamin, led to higher eicosapentaenoic acid (EPA) composition at endpoint. Docosahexaenoic acid (DHA) composition was unchanged following treatment. The long chain LC n-3 PUFA index was only higher, compared to placebo, in the group receiving the combination of 6 g of fish oil and the multivitamin. Analysis by gender revealed that all treatments increased EPA incorporation in females while, in males, EPA was only significantly increased by the 6 g fish oil multivitamin combination. There was considerable individual variability in the red blood cell incorporation of EPA and DHA at endpoint. Gender contributed to a large proportion of this variability with females generally showing higher LC n-3 PUFA composition at endpoint. In conclusion, the incorporation of LC n-3 PUFA into red blood cells was influenced by dosage, the concurrent intake of vitamin/minerals and gender.

Predictors of treatment response in young people at ultra-high risk for psychosis who received long-chain omega-3 fatty acids.

Previous efforts in the prospective evaluation of individuals who experience attenuated psychotic symptoms have attempted to isolate mechanisms underlying the onset of full-threshold psychotic illness. In contrast, there has been little research investigating specific predictors of positive outcomes. In this study, we sought to determine biological and clinical factors associated with treatment response, here indexed by functional improvement in a pre-post examination of a 12-week randomized controlled intervention in individuals at ultra-high risk (UHR) for psychosis. Participants received either long-chain omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) or placebo. To allow the determination of factors specifically relevant to each intervention, and to be able to contrast them, both treatment groups were investigated in parallel. Univariate linear regression analysis indicated that higher levels of erythrocyte membrane α-linolenic acid (ALA; the parent fatty acid of the ω-3 family) and more severe negative symptoms at baseline predicted subsequent functional improvement in the treatment group, whereas less severe positive symptoms and lower functioning at baseline were predictive in the placebo group. A multivariate machine learning analysis, known as Gaussian Process Classification (GPC), confirmed that baseline fatty acids predicted response to treatment in the ω-3 PUFA group with high levels of sensitivity, specificity and accuracy. In addition, GPC revealed that baseline fatty acids were predictive in the placebo group. In conclusion, our investigation indicates that UHR patients with higher levels of ALA may specifically benefit from ω-3 PUFA supplementation. In addition, multivariate machine learning analysis suggests that fatty acids could potentially be used to inform prognostic evaluations and treatment decisions at the level of the individual. Notably, multiple statistical analyses were conducted in a relatively small sample, limiting the conclusions that can be drawn from what we believe to be a first-of-its-kind study. Additional studies with larger samples are therefore needed to evaluate the generalizability of these findings.

Fishy business : effect of omega-3 fatty acids on zinc transporters and free zinc availability in human neuronal cells

Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain, DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total. DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on. On the other hand, zinc is the most abundant trace metal in the human brain. There are many scientific studies linking zinc, especially excess amounts of free zinc, to cellular death. Neurodegenerative diseases, such as Alzheimer's disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between omega-3 fatty acids, zinc transporter levels and free zinc availability at cellular levels. Many other studies have also suggested a possible omega-3 and zinc effect on neurodegeneration and cellular death. Therefore, in this review, we will examine the effect of omega-3 fatty acids on zinc transporters and the importance of free zinc for human neuronal cells. Moreover, we will evaluate the collective understanding of mechanism(s) for the interaction of these elements in neuronal research and their significance for the diagnosis and treatment of neurodegeneration.

Short-chain fatty acids increase expression and secretion of stromal cell-derived factor-1 in mouse and human pre-adipocytes

Objective: Stromal cell-derived factor-1 (SDF-1) is expressed in pre-adipocytes but its role is unknown. We investigated butyrate (a histone deacetylase inhibitor - HDACi) and other short-chain fatty acids (SCFA) in the regulation of SDF-1. We further investigated whether effects of SCFA were signalled through G protein-coupled receptors FFA2 and FFA3. Design and Results: SDF-1 mRNA expression and protein secretion were studied in 3T3-L1 cells and human pre-adipocytes. SDF-1 was abundant, with mRNA and protein levels increased by butyrate. This was replicated with acetate and propionate, but not with trichostatin or valproate. Trichostatin inhibited SDF-1 secretion. Pertussis toxin blocked stimulation by butyrate. The order of potency of SCFA in stimulating SDF-1 (C3 > C4 > C2) is consistent with action through FFA3. Silencing the FFA3 gene abolished butyrate-stimulated SDF-1 expression and secretion. FFA3 was expressed in both pre-adipocytes and adipocytes, while FFA2 was expressed in adipocytes only. SDF-1 expression was low in murine macrophage J774.2 cells, while the SDF-1 receptor CXCR4 was absent from 3T3-L1 cells but abundant in J774.2 macrophages. In human pre-adipocytes, FFA3 was also expressed and SCFA increased SDF-1 secretion. Conclusions: SDF-1 and CXCR4 may mediate the interaction between adipose stromal cells and macrophages. Effects of SCFA are mediated through FFA3, but not histone deacetylase inhibition.

Experimental reduction in dietary omega-3 polyunsaturated fatty acids depresses sperm competitiveness

 The health benefits of diets containing rich sources of long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA) are well documented and include reductions in the risk of several diseases typical of Western societies. The dietary intake of n-3 LC-PUFA has also been linked to fertility, and there is abundant evidence that a range of ejaculate traits linked to fertility in humans, livestock and other animals depend on an adequate intake of n-3 LC-PUFA from dietary sources. However, relatively few studies have explored how n-3 LC-PUFA influence reproductive fitness, particularly in the context of sexual selection. Here, we show that experimental reduction in the level of n-3 LC-PUFA in the diet of guppies (Poecilia reticulata) depresses a male’s share of paternity when sperm compete for fertilization, confirming that the currently observed trend for reduced n-3 LC-PUFA in western diets has important implications for individual reproductive fitness.