Early to sustained impacts of lethal radiation on circulating miRNAs in a minipig model.

Early diagnosis of lethal radiation is imperative since its intervention time windows are considerably short. Hence, ideal diagnostic candidates of radiation should be easily accessible, enable to inform about the stress history and objectively triage subjects in a time-efficient manner. Therefore, the small molecules such as metabolites and microRNAs (miRNAs) from plasma are legitimate biomarker candidate for lethal radiation. Our objectives were to comprehend the radiation-driven molecular pathogenesis and thereby determine biomarkers of translational potential. We investigated an established minipig model of LD70/45 total body irradiation (TBI). In this pilot study, plasma was collected pre-TBI and at multiple time points post-TBI. The majority of differentially expressed miRNAs and metabolites were perturbed immediately after TBI that potentially underlined the severity of its acute impact. The integrative network analysis of miRNA and metabolites showed a cohesive response; the early and consistent perturbations of networks were linked to cancer and the shift in musculoskeletal atrophy synchronized with the comorbidity-networks associated with inflammation and bioenergy synthesis. Subsequent comparative pipeline delivered 92 miRNAs, which demonstrated sequential homology between human and minipig, and potentially similar responses to lethal radiation across these two species. This panel promised to retrospectively inform the time since the radiation occurred; thereby could facilitate knowledge-driven interventions.

Systemic effects of BMP2 treatment of fractures on non-injured skeletal sites during spaceflight.

Unloading associated with spaceflight results in bone loss and increased fracture risk. Bone morphogenetic protein 2 (BMP2) is known to enhance bone formation, in part, through molecular pathways associated with mechanical loading; however, the effects of BMP2 during spaceflight remain unclear. Here, we investigated the systemic effects of BMP2 on mice sustaining a femoral fracture followed by housing in spaceflight (International Space Station or ISS) or on Earth. We hypothesized that in spaceflight, the systemic effects of BMP2 on weight-bearing bones would be blunted compared to that observed on Earth. Nine-week-old male mice were divided into four groups: 1) Saline+Earth; 2) BMP+Earth; 3) Saline+ISS; and 4) BMP+ISS (n = 10 mice/group, but only n = 5 mice/group were reserved for micro-computed tomography analyses). All mice underwent femoral defect surgery and were followed for approximately 4 weeks. We found a significant reduction in trabecular separation within the lumbar vertebrae after administering BMP2 at the fracture site of mice housed on Earth. In contrast, BMP2 treatment led to a significant increase in trabecular separation concomitant with a reduction in trabecular number within spaceflown tibiae. Although these and other lines of evidence support our hypothesis, the small sample size associated with rodent spaceflight studies limits interpretations. That said, it appears that a locally applied single dose of BMP2 at the femoral fracture site can have a systemic impact on distant bones, affecting bone quantity in several skeletal sites. Moreover, our results suggest that BMP2 treatment works through a pathway involving mechanical loading in which the best outcomes during its treatment on Earth occurred in the weight-bearing bones and in spaceflight occurred in bones subjected to higher muscle contraction.

Predicting fracture healing with blood biomarkers: the potential to assess patient risk of fracture nonunion.

Fracture non-union is a significant orthopaedic problem affecting a substantial number of patients yearly. Treatment of nonunions is devastating to patients and costly to the healthcare system. Unfortunately, the diagnosis of non-union is typically made in a reactionary fashion by an orthopaedic surgeon based on clinical assessment and radiographic features several months into treatment. For this reason, investigators have been trying to develop prediction algorithms; however, these have relied on population-based approaches and lack the predictive capability necessary to make individual treatment decisions. There is also a growing body of literature focussed on identifying blood biomarkers that are associated with non-union. This review describes the research that has been done in this area. Further studies of patient-centered, precision medicine approaches will likely improve fracture non-union diagnostic/prognostic capabilities.

Gene-metabolite networks associated with impediment of bone fracture repair in spaceflight.

Adverse effects of spaceflight on musculoskeletal health increase the risk of bone injury and impairment of fracture healing. Its yet elusive molecular comprehension warrants immediate attention, since space travel is becoming more frequent. Here we examined the effects of spaceflight on bone fracture healing using a 2 mm femoral segmental bone defect (SBD) model. Forty, 9-week-old, male C57BL/6J mice were randomized into 4 groups: 1) Sham surgery on Ground (G-Sham); 2) Sham surgery housed in Spaceflight (FLT-Sham); 3) SBD surgery on Ground (G-Surgery); and 4) SBD surgery housed in Spaceflight (FLT-Surgery). Surgery procedures occurred 4 days prior to launch; post-launch, the spaceflight mice were house in the rodent habitats on the International Space Station (ISS) for approximately 4 weeks before euthanasia. Mice remaining on the Earth were subjected to identical housing and experimental conditions. The right femur from half of the spaceflight and ground groups was investigated by micro-computed tomography (µCT). In the remaining mice, the callus regions from surgery groups and corresponding femoral segments in sham mice were probed by global transcriptomic and metabolomic assays. µCT confirmed escalated bone loss in FLT-Sham compared to G-Sham mice. Comparing to their respective on-ground counterparts, the morbidity gene-network signal was inhibited in sham spaceflight mice but activated in the spaceflight callus. µCT analyses of spaceflight callus revealed increased trabecular spacing and decreased trabecular connectivity. Activated apoptotic signals in spaceflight callus were synchronized with inhibited cell migration signals that potentially hindered the wound site to recruit growth factors. A major pro-apoptotic and anti-migration gene network, namely the RANK-NFκB axis, emerged as the central node in spaceflight callus. Concluding, spaceflight suppressed a unique biomolecular mechanism in callus tissue to facilitate a failed regeneration, which merits a customized intervention strategy.

Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling.

Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9-2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries.

Analysis of the effects of spaceflight and local administration of thrombopoietin to a femoral defect injury on distal skeletal sites.

With increased human presence in space, bone loss and fractures will occur. Thrombopoietin (TPO) is a recently patented bone healing agent. Here, we investigated the systemic effects of TPO on mice subjected to spaceflight and sustaining a bone fracture. Forty, 9-week-old, male, C57BL/6 J were divided into 4 groups: (1) Saline+Earth; (2) TPO + Earth; (3) Saline+Flight; and (4) TPO + Flight (n = 10/group). Saline- and TPO-treated mice underwent a femoral defect surgery, and 20 mice were housed in space ("Flight") and 20 mice on Earth for approximately 4 weeks. With the exception of the calvarium and incisor, positive changes were observed in TPO-treated, spaceflight bones, suggesting TPO may improve osteogenesis in the absence of mechanical loading. Thus, TPO, may serve as a new bone healing agent, and may also improve some skeletal properties of astronauts, which might be extrapolated for patients on Earth with restraint mobilization and/or are incapable of bearing weight on their bones.

Gene-Metabolite Network Linked to Inhibited Bioenergetics in Association With Spaceflight-Induced Loss of Male Mouse Quadriceps Muscle.

Prolonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of the National Aeronautics and Space Administration (NASA), we assayed the large-scale mRNA and metabolomic perturbations in the quadriceps of C57BL/6j male mice that lived in spaceflight (FLT) or on the ground (control or CTR) for approximately 4 weeks. The wet weights of the quadriceps were significantly reduced in FLT mice. Next-generation sequencing and untargeted mass spectroscopic assays interrogated the gene-metabolite landscape of the quadriceps. A majority of top-ranked differentially suppressed genes in FLT encoded proteins from the myosin or troponin families, suggesting sarcomere alterations in space. Significantly enriched gene-metabolite networks were found linked to sarcomeric integrity, immune fitness, and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of mitochondrial DNA copy numbers in FLT mice underlined the energy deprivation associated with spaceflight-induced stress. This hypothesis was reinforced by the transcriptomic sequencing-metabolomics integrative analysis that showed inhibited networks related to protein, lipid, and carbohydrate metabolism, and adenosine triphosphate (ATP) synthesis and hydrolysis. Finally, we discovered important upstream regulators, which could be targeted for next-generation therapeutic intervention for chronic disuse of the musculoskeletal system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

microRNA and Metabolite Signatures Linked to Early Consequences of Lethal Radiation.

Lethal total body irradiation (TBI) triggers multifactorial health issues in a potentially short time frame. Hence, early signatures of TBI would be of great clinical value. Our study aimed to interrogate microRNA (miRNA) and metabolites, two biomolecules available in blood serum, in order to comprehend the immediate impacts of TBI. Mice were exposed to a lethal dose (9.75 Gy) of Cobalt-60 gamma radiation and euthanized at four time points, namely, days 1, 3, 7 and 9 post-TBI. Serum miRNA libraries were sequenced using the Illumina small RNA sequencing protocol, and metabolites were screened using a mass spectrometer. The degree of early impacts of irradiation was underscored by the large number of miRNAs and metabolites that became significantly expressed during the Early phase (day 0 and 1 post-TBI). Radiation-induced inflammatory markers for bone marrow aplasia and pro-sepsis markers showed early elevation with longitudinal increment. Functional analysis integrating miRNA-protein-metabolites revealed inflammation as the overarching host response to lethal TBI. Early activation of the network linked to the synthesis of reactive oxygen species was associated with the escalated regulation of the fatty acid metabolism network. In conclusion, we assembled a list of time-informed critical markers and mechanisms of significant translational potential in the context of a radiation exposure event.

The effects of spaceflight and fracture healing on distant skeletal sites.

Spaceflight results in reduced mechanical loading of the skeleton, which leads to dramatic bone loss. Low bone mass is associated with increased fracture risk, and this combination may compromise future, long-term, spaceflight missions. Here, we examined the systemic effects of spaceflight and fracture surgery/healing on several non-injured bones within the axial and appendicular skeleton. Forty C57BL/6, male mice were randomized into the following groups: (1) Sham surgery mice housed on the earth (Ground + Sham); (2) Femoral segmental bone defect surgery mice housed on the earth (Ground + Surgery); (3) Sham surgery mice housed in spaceflight (Flight + Sham); and (4) Femoral segmental bone defect surgery mice housed in spaceflight (Flight + Surgery). Mice were 9 weeks old at the time of launch and were euthanized approximately 4 weeks after launch. Micro-computed tomography (µCT) was used to evaluate standard bone parameters in the tibia, humerus, sternebra, vertebrae, ribs, calvarium, mandible, and incisor. One intriguing finding was that both spaceflight and surgery resulted in virtually identical losses in tibial trabecular bone volume fraction, BV/TV (24-28% reduction). Another important finding was that surgery markedly changed tibial cortical bone geometry. Understanding how spaceflight, surgery, and their combination impact non-injured bones will improve treatment strategies for astronauts and terrestrial humans alike.

Forces associated with launch into space do not impact bone fracture healing.

Segmental bone defects (SBDs) secondary to trauma invariably result in a prolonged recovery with an extended period of limited weight bearing on the affected limb. Soldiers sustaining blast injuries and civilians sustaining high energy trauma typify such a clinical scenario. These patients frequently sustain composite injuries with SBDs in concert with extensive soft tissue damage. For soft tissue injury resolution and skeletal reconstruction a patient may experience limited weight bearing for upwards of 6 months. Many small animal investigations have evaluated interventions for SBDs. While providing foundational information regarding the treatment of bone defects, these models do not simulate limited weight bearing conditions after injury. For example, mice ambulate immediately following anesthetic recovery, and in most cases are normally ambulating within 1-3 days post-surgery. Thus, investigations that combine disuse with bone healing may better test novel bone healing strategies. To remove weight bearing, we have designed a SBD rodent healing study in microgravity (µG) on the International Space Station (ISS) for the Rodent Research-4 (RR-4) Mission, which launched February 19, 2017 on SpaceX CRS-10 (Commercial Resupply Services). In preparation for this mission, we conducted an end-to-end mission simulation consisting of surgical infliction of SBD followed by launch simulation and hindlimb unloading (HLU) studies. In brief, a 2 mm defect was created in the femur of 10 week-old C57BL6/J male mice (n = 9-10/group). Three days after surgery, 6 groups of mice were treated as follows: 1) Vivarium Control (maintained continuously in standard cages); 2) Launch Negative Control (placed in the same spaceflight-like hardware as the Launch Positive Control group but were not subjected to launch simulation conditions); 3) Launch Positive Control (placed in spaceflight-like hardware and also subjected to vibration followed by centrifugation); 4) Launch Positive Experimental (identical to Launch Positive Control group, but placed in qualified spaceflight hardware); 5) Hindlimb Unloaded (HLU, were subjected to HLU immediately after launch simulation tests to simulate unloading in spaceflight); and 6) HLU Control (single housed in identical HLU cages but not suspended). Mice were euthanized 28 days after launch simulation and bone healing was examined via micro-Computed Tomography (µCT). These studies demonstrated that the mice post-surgery can tolerate launch conditions. Additionally, forces and vibrations associated with launch did not impact bone healing (p = .3). However, HLU resulted in a 52.5% reduction in total callus volume compared to HLU Controls (p = .0003). Taken together, these findings suggest that mice having a femoral SBD surgery tolerated the vibration and hypergravity associated with launch, and that launch simulation itself did not impact bone healing, but that the prolonged lack of weight bearing associated with HLU did impair bone healing. Based on these findings, we proceeded with testing the efficacy of FDA approved and novel SBD therapies using the unique spaceflight environment as a novel unloading model on SpaceX CRS-10.

Metabolomic analyses reveal lipid abnormalities and hepatic dysfunction in non-human primate model for Yersinia pestis.

INTRODUCTION: Pneumonic plague is caused by the aerosolized form of Yersinia pestis and is a highly virulent infection with complex clinical consequences, and without treatment, the fatality rate approaches 100%. The exact mechanisms of disease progression are unclear, with limited work done using metabolite profiling to study disease progression. OBJECTIVE: The aim of this pilot study was to profile the plasma metabolomics in an animal model of Y. pestis infection. METHODS: In this study, African Green monkeys were challenged with the highly virulent, aerosolized Y. pestis strain CO92, and untargeted metabolomics profiling of plasma was performed using liquid and gas chromatography with mass spectrometry. RESULTS: At early time points post-exposure, we found significant increases in polyunsaturated, long chain fatty acid metabolites with p values ranging from as low as 0.000001 (ratio = 1.94) for the metabolite eicosapentaenoate to 0.04 (ratio = 1.36) for the metabolite adrenate when compared to time-matched controls. Multiple acyl carnitines metabolites were increased at earlier time points and could be a result of fatty acid oxidation defects with p values ranging from as low as 0.00001 (ratio = 2.95) for the metabolite octanoylcarnitine to 0.04 (ratio = 1.33) for metabolite deoxycarnitine when compared to time-matched controls. Dicarboxylic acids are important metabolic products of fatty acids oxidation, and when compared to time matched controls, were higher at earlier time points where metabolite tetradecanedioate has a ratio of 4.09 with significant p value of 0.000002 and adipate with a ratio of 1.12 and p value of 0.004. The metabolites from lysolipids (with significant p values ranging from 0.00006 for 1-oleoylglycerophosphoethanolamine to 0.04 for 1-stearoylglycerophosphoethanolamine and a ratio of 0.47 and 0.78, respectively) and bile acid metabolism (with significant p values ranging from 0.02 for cholate to 0.04 for deoxycholate and a ratio of 0.39 and 0.66, respectively) pathways were significantly lower compared to their time-matched controls during the entire course of infection. Metabolite levels from amino acid pathways were disrupted, and a few from the leucine, isoleucine and valine pathway were significantly higher (p values ranging from 0.002 to 0.04 and ratios ranging from 1.3 to 1.5, respectively), whereas metabolites from the urea cycle, arginine and proline pathways were significantly lower (p values ranging from 0.00008 to 0.02 and ratios ranging from 0.5 to 0.7, respectively) during the course of infection. CONCLUSIONS: The involvement of several lipid pathways post-infection suggested activation of pathways linked to inflammation and oxidative stress. Metabolite data further showed increased energy demand, and multiple metabolites indicated potential hepatic dysfunction. Integration of blood metabolomics and transcriptomics data identified linoleate as a core metabolite with cross-talk with multiple genes from various time points. Collectively, the data from this study provided new insights into the mechanisms of Y. pestis pathogenesis that may aid in development of therapeutics.

Changes in intestinal microbiota composition and metabolism coincide with increased intestinal permeability in young adults under prolonged physiological stress.

The magnitude, temporal dynamics, and physiological effects of intestinal microbiome responses to physiological stress are poorly characterized. This study used a systems biology approach and a multiple-stressor military training environment to determine the effects of physiological stress on intestinal microbiota composition and metabolic activity, as well as intestinal permeability (IP). Soldiers (n = 73) were provided three rations per day with or without protein- or carbohydrate-based supplements during a 4-day cross-country ski-march (STRESS). IP was measured before and during STRESS. Blood and stool samples were collected before and after STRESS to measure inflammation, stool microbiota, and stool and plasma global metabolite profiles. IP increased 62 ± 57% (mean ± SD, P < 0.001) during STRESS independent of diet group and was associated with increased inflammation. Intestinal microbiota responses were characterized by increased α-diversity and changes in the relative abundance of >50% of identified genera, including increased abundance of less dominant taxa at the expense of more dominant taxa such as Bacteroides Changes in intestinal microbiota composition were linked to 23% of metabolites that were significantly altered in stool after STRESS. Together, pre-STRESS Actinobacteria relative abundance and changes in serum IL-6 and stool cysteine concentrations accounted for 84% of the variability in the change in IP. Findings demonstrate that a multiple-stressor military training environment induced increases in IP that were associated with alterations in markers of inflammation and with intestinal microbiota composition and metabolism. Associations between IP, the pre-STRESS microbiota, and microbiota metabolites suggest that targeting the intestinal microbiota could provide novel strategies for preserving IP during physiological stress.NEW & NOTEWORTHY Military training, a unique model for studying temporal dynamics of intestinal barrier and intestinal microbiota responses to stress, resulted in increased intestinal permeability concomitant with changes in intestinal microbiota composition and metabolism. Prestress intestinal microbiota composition and changes in fecal concentrations of metabolites linked to the microbiota were associated with increased intestinal permeability. Findings suggest that targeting the intestinal microbiota could provide novel strategies for mitigating increases in intestinal permeability during stress.

Characterization of the interaction of staphylococcal enterotoxin B with CD1d expressed in human renal proximal tubule epithelial cells.

BACKGROUND: Participation of renal cells in the pathogenesis of staphylococcal enterotoxin B (SEB) is critical for late cleansing and sequestration of the antigens facilitated by CD1d mediated antigen sensing and recognition. This is a noted deviation from the typical antigen recognition process that recruits the major histocompatibility complex class II (MHC II) molecules. The immunological importance of CD1d is underscored by its influences on the performances of natural killer T-cells and thereby mediates the innate and adaptive immune systems. RESULTS: Using diffraction-based dotReady™ immunoassays, the present study showed that SEB directly and specifically conjugated to CD1d. The specificity of the conjugation between SEB and CD1d expressed on human renal proximal tubule epithelial cells (RPTEC) was further established by selective inhibition of CD1d prior to its exposure to SEB. We found that SEB induced the expression of CD1d on the cell surface prompting a rapid conjugation between them. The mRNA transcripts encoding CD1d remained elevated potentially after completing the antigen cleansing process. CONCLUSION: Molecular targets associated with the delayed pathogenic response have essential therapeutic values. Particularly in the event of bioterrorism, the caregivers are typically able to intervene much later than the toxic exposures. Given circumstances mandate a paradigm shift from the conventional therapeutic strategy that counts on targeting the host markers responding to the early assault of pathogens. We demonstrated the role of CD1d in the late stage of pathogen recognition and cleansing, and thereby underscored its clinical potential in treating bioweaponizable antigens, such as Staphylococcal enterotoxin B (SEB).

Transcriptomic analysis of the effects of a fish oil enriched diet on murine brains.

The health benefits of fish oil enriched with high omega-3 polyunsaturated fatty acids (n-3 PUFA) are widely documented. Fish oil as dietary supplements, however, show moderate clinical efficacy, highlighting an immediate scope of systematic in vitro feedback. Our transcriptomic study was designed to investigate the genomic shift of murine brains fed on fish oil enriched diets. A customized fish oil enriched diet (FD) and standard lab diet (SD) were separately administered to two randomly chosen populations of C57BL/6J mice from their weaning age until late adolescence. Statistical analysis mined 1,142 genes of interest (GOI) differentially altered in the hemibrains collected from the FD- and SD-fed mice at the age of five months. The majority of identified GOI (∼ 40%) encodes proteins located in the plasma membrane, suggesting that fish oil primarily facilitated the membrane-oriented biofunctions. FD potentially augmented the nervous system's development and functions by selectively stimulating the Src-mediated calcium-induced growth cascade and the downstream PI3K-AKT-PKC pathways. FD reduced the amyloidal burden, attenuated oxidative stress, and assisted in somatostatin activation-the signatures of attenuation of Alzheimer's disease, Parkinson's disease, and affective disorder. FD induced elevation of FKBP5 and suppression of BDNF, which are often linked with the improvement of anxiety disorder, depression, and post-traumatic stress disorder. Hence we anticipate efficacy of FD in treating illnesses such as depression that are typically triggered by the hypoactivities of dopaminergic, adrenergic, cholinergic, and GABAergic networks. Contrastingly, FD's efficacy could be compromised in treating illnesses such as bipolar disorder and schizophrenia, which are triggered by hyperactivities of the same set of neuromodulators. A more comprehensive investigation is recommended to elucidate the implications of fish oil on disease pathomechanisms, and the result-driven repositioning of fish oil utilization may revitalize its therapeutic efficacy.

Prosthodontic rehabilitation of velopharyngeal disorders-a case series.

Acquired palatal defects results from cancer resection, whereas cleft palate is the main cause of congenital defects. Both these condition leads to impaired speech intelligibility, seepage of nasal secretions into the oral cavity and vice versa while difficulty in swallowing and hyper nasality is also eminent. In this case report three different technique for treating velopharyngeal disorders have been described.

Differential effects of omega-3 and omega-6 Fatty acids on gene expression in breast cancer cells.

Essential fatty acids have long been identified as possible oncogenic factors. Existing reports suggest omega-6 (omega-6) essential fatty acids (EFA) as pro-oncogenic and omega-3 (omega-3) EFA as anti-oncogenic factors. The omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), inhibit the growth of human breast cancer cells while the omega-6 fatty acids induces growth of these cells in animal models and cell lines. In order to explore likely mechanisms for the modulation of breast cancer cell growth by omega-3 and omega-6 fatty acids, we examined the effects of arachidonic acid (AA), linoleic acid (LA), EPA and DHA on human breast cancer cell lines using cDNA microarrays and quantitative polymerase chain reaction. MDA-MB-231, MDA-MB-435s, MCF-7 and HCC2218 cell lines were treated with the selected fatty acids for 6 and 24 h. Microarray analysis of gene expression profiles in the breast cancer cells treated with both classes of fatty acids discerned essential differences among the two classes at the earlier time point. The differential effects of omega-3 and omega-6 fatty acids on the breast cancer cells were lessened at the late time point. Data mining and statistical analyses identified genes that were differentially expressed between breast cancer cells treated with omega-3 and omega-6 fatty acids. Ontological investigations have associated those genes to a broad spectrum of biological functions, including cellular nutrition, cell division, cell proliferation, metastasis and transcription factors etc., and thus presented an important pool of biomarkers for the differential effect of omega-3 and omega-6EFAs.

Expression patterns of fatty acid binding proteins in breast cancer cells.

We studied the expression levels of fatty acid binding proteins in breast normal and cancer cell lines. Liver fatty acid binding protein (L-FABP) and intestine fatty acid binding proteins were shown to be up-regulated in breast cancer cell lines while adipose- and epidermal-fatty acid binding proteins were down regulated in breast cancer cells compared to normal breast cell lines. We have previously shown that blocking the expression of L-FABP resulted in remarkable effects on apoptosis and cell proliferation of prostate cancer cell lines (Hammamieh et al 2004). To study the mechanism of effect of the liver fatty acid binding protein in breast cancer cells, we designed an antisense oligodeoxynucleotide to block the production of liver-FABP in MCF-7 cells. The antisense was shown to inhibit the expression of L-FABP and to induce apoptosis in MCF-7 cells. This study examines the mechanism by which L-FABP antisense regulates proliferation and apoptosis in breast cancer cell lines. We used human cDNA microarrays to explore differentially expressed genes in MCF-7 breast cancer cells treated with L-FABP antisense oligonucleotide. Some of the genes that were differentially expressed were confirmed using quantitative RT-PCR. Genes related to cell growth, proliferation and angiogenesis showed significant variations. This suggests a possible use of these antisense ODNs as therapeutic agents for breast cancer in future.

Molecular impacts of antisense complementary to the liver fatty acid binding protein (FABP) mRNA in DU 145 prostate cancer cells in vitro.

Previous studies in our laboratory have showed that liver (L)-FABP and intestine-FABP were upregulated in prostate cancer cells whereas Adipose-FABP and epidermal-FABP were down regulated in cancer cells when compared to normal cells in tissue cultures and biopsy samples (Das et al, 2001). We have also shown that blocking the expression of L-FABP resulted in remarkable effects on apoptosis and cell proliferation of prostate cancer cell lines. Another study showed that induction of A-FABP inhibited proliferation DU 145 prostate cancer cells (DeSantis et. al., Journal of Experimental Therapeutics and Oncology, 4:91-100, 2004) and caused a downregulation of the L-FABP transcript levels. This suggests that there is a correlation in the expression between of A-FABP and L-FABP in prostate cancer cells and that high expression of A-FABP is associated with a downregulation of L-FABP and vice versa. This study examines the mechanism by which L-FABP antisense regulates proliferation and apoptosis in prostate cancer cell lines. We used human cDNA array blots and custom DNA microarrays to explore differentially expressed genes in DU 145 prostate cancer cells treated with L-FABP antisense oligonucleotide. Genes that were differentially expressed were confirmed using quantitative RT-PCR. Genes correlating with proliferation were downregulated and antiproliferative genes were upregulated in response to antisense to L-FABP. This suggests a possible use of these antisense ODNs as therapeutic agents for prostate cancer in future.