Cross-species conservation of a transposase-linked element enables genetic sexing of commercial populations of Russian sturgeon (Acipenser gueldenstaedtii).

All-female culture of sturgeon is essential for efficient caviar production. However, Russian sturgeon (Acipenser gueldenstaedtii) does not exhibit external sexual dimorphism, and therefore, commercial farms apply gonadal endoscopy or ultrasound at the earliest age of 4-5 years to separate the sexes, with ~90% accuracy. Recently, a dominant genomic marker (AllWSEX2) has been found with association to femaleness in sturgeons. We developed a duplex PCR (dAllWSEX2) with the adjacent bmp7 gene as an internal control, to validate an effective PCR. Robust amplification of control fragments was observed for all samples of our commercial A. gueldenstaedtii stock (n = 337). The dAllWSEX2 assay was significantly associated with sex (n = 43, p < 1.6 × 10-8 ), yet four (18%) of the endoscopy-determined females were genetic males. To examine whether some females display a male genetic profile, we tested 96 egg-producing females, which were all verified as genetic females, indicating that the observed mismatches may be attributed to wrong sexing by endoscopy. Application of dAllWSEX2 on 100 7-month-old fish showed no sex-dependent differences in body weight, indicating that weighing is not an applicable tool for sorting females at a young age. Sanger sequencing of the bmp7 fragment revealed octaploidy and sex-independent variation, suggesting that the critical sex-determining region harboring AllWSEX2 is small. In keeping with a model of a single-ploidy encoding female determination, AllWSEX2 showed no variation despite being a transposase-linked repetitive element. Cross-species conservation of AllWSEX2, and absence of annotated sex-determination genes in this region suggests that, in sturgeons, the sex-determining mechanism is different from mechanisms identified in other fish.

Whole-Genome Inter-Sex Variation in Russian Sturgeon (Acipenser gueldenstaedtii).

The Russian sturgeon (Acipenser gueldenstaedtii, AG) is an endangered fish species increasingly raised on fish farms for black caviar. Understanding the process of sex determination in AG is, therefore, of scientific and commercial importance. AG lacks sexual dimorphism until sexual maturation and has a predominantly octoploid genome without a definite sex chromosome. A conserved short female-specific genomic sequence was recently described, leading to the development of a genetic sex marker. However, no biological function has been reported for this sequence. Thus, the mechanism of sex determination and the overall inter-sex genomic variation in AG are still unknown. To comprehensively analyze the inter-sex genomic variation and assess the overall inter-species variation between AG and A. ruthenus (AR, sterlet), a related tetraploid sturgeon species, we performed whole-genome sequencing on DNA from 10 fish-farm-raised adult AG (5 males and 5 females). We produced a partially assembled, ~2390 MBp draft genome for AG. We validated in AG the female-specific region previously described in AR. We identified ~2.8 million loci (SNP/indels) varying between the species, but only ~7400 sex-associated loci in AG. We mapped the sex-associated AG loci to the AR genome and identified 15 peaks of sex-associated variation (10 kb segments with 30 or more sex-associated variants), 1 of which matched the previously reported sex-variable region. Finally, we identified 14 known and predicted genes in proximity to these peaks. Our analysis suggests that one or more of these genes may have functional roles in sex determination and/or sexual differentiation in sturgeons. Further functional studies are required to elucidate these roles.

Natural Products Targeting Cancer Stem Cells for Augmenting Cancer Therapeutics.

Cancer stem cells (CSC) have been identified in several types of solid tumors. In some cases, CSC may be the source of all the tumor cells, the cause of the tumor's resistance to chemotherapeutic agents, and the source of metastatic cells. Thus, a combination therapy targeting non-CSC tumor cells as well as specifically targeting CSCs holds the potential to be highly effective. Natural products (NPs) have been a historically rich source of biologically active compounds and are known for their ability to influence multiple signaling pathways simultaneously with negligible side effects. In this review, we discuss the potential of NPs in targeting multiple signaling pathways in CSC and their potential to augment the efficacy of standard cancer therapy. Specifically, we focus on the anti-CSC activities of flavonoids, FDA-approved drugs originating from natural sources. Additionally, we emphasize the potential of NPs in targeting microRNA-mediated signaling, given the roles of microRNA in the maintenance of the CSC phenotype.

Obesity impacts the regulation of miR-10b and its targets in primary breast tumors.

BACKGROUND: Obesity increases breast cancer (BC) risk in post-menopausal women by mostly unknown molecular mechanisms which may partly be regulated by microRNAs (miRNAs). METHODS: We isolated RNA from paired benign and malignant biopsies from 83 BC patients and determined miRNA profiles in samples from 12 women at the extremes of the BMI distribution by RNA-seq. Candidates were validated in all samples. Associations between miR-10b expression and validated target transcript levels, and effects of targeted manipulation of miR-10b levels in a primary BC cell line on proliferation and invasion potential, were explored. RESULTS: Of the 148 miRNAs robustly expressed in breast tissues, the levels of miR-21, miR-10b, miR-451a, miR-30c, and miR-378d were significantly associated with presence of cancer. Of these, miR-10b showed a stronger down-regulation in the tumors of the obese subjects, as opposed to the lean. In ductal but not lobular tumors, significant inverse correlations were observed between the tumor levels of miR-10b and miR-30c and the mRNA levels of cancer-relevant target genes SRSF1, PIEZO1, MAPRE1, CDKN2A, TP-53 and TRA2B, as well as tumor grade. Suppression of miR-10b levels in BT-549 primary BC-derived cells increased cell proliferation and invasive capacity, while exogenous miR-10b mimic decreased invasion. Manipulation of miR-10b levels also inversely affected the mRNA levels of miR-10b targets BCL2L11, PIEZO1 and NCOR2. CONCLUSIONS: Our findings suggest that miR-10b may be a mediator between obesity and cancer in post-menopausal women, regulating several known cancer-relevant genes. MiR-10b expression may have diagnostic and therapeutic implications for the incidence and prognosis of BC in obese women.

Evaluating Azoxystrobin Seed Coating Against Maize Late Wilt Disease Using a Sensitive qPCR-Based Method.

Harpophora maydis, a phytopathogenic fungus, causes late wilt, a severe vascular maize disease characterized by relatively rapid wilting of maize plants near fertilization. The disease is currently controlled using resistant varieties. Here, we evaluated seed coating efficiency with azoxystrobin against H. maydis in a series of in vitro and in vivo trials. A real-time polymerase chain reaction (qPCR)-based method was developed and proved to be a sensitive, accurate tool for monitoring H. maydis DNA inside infected seeds, sprouts, and tissues of mature plants. In the early growth stages, the chemical coating drastically reduced the pathogen DNA prevalence in host tissues and minimized the suppressing effect on the plants' biomass and development. In an infested field, the qPCR assay identified the pathogen 20 days after seeding, up to a month before conventional PCR detection. In the resistant fodder maize cultivar 32D99, which showed only minor disease symptoms, the seed coating blocked fungal progression and increased cob and plant weight by 39 and 60%, respectively. Nevertheless, this treatment was unable to protect a sensitive maize hybrid, cultivar Prelude, at the disease wilting breakout (60 days after sowing). These results encourage further examination of azoxystrobin and other fungicides in the field using the qPCR detection method to evaluate their efficiency.

MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase.

MicroRNAs (miRNAs) contribute to both neuronal and immune cell fate, but their involvement in intertissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induced leukocyte overexpression of the AChE-targeting miR-132. Injected locked nucleic acid (LNA)-modified anti-miR-132 oligonucleotide depleted miR-132 amounts while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3'UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice overexpressing 3'UTR null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 upregulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening avenues for study and therapeutic manipulations of the neuro-immune dialog.

Elevated Levels of miR-122 in Serum May Contribute to Improved Endothelial Function and Lower Oncologic Risk Following Bariatric Surgery.

BACKGROUND: Weight loss surgery is the most effective treatment for obesity, and it reduces cardiovascular and cancer risk through poorly understood mechanisms. MicroRNAs (miRNAs) are short RNA molecules that regulate the stability and translation of many mRNAs. We hypothesized that levels of specific circulating miRNAs are altered following surgery and may contribute to lower cancer risk. OBJECTIVES: To investigate the change of miRNA following surgery. METHODS: All patients underwent gastric "sleeve operation". RNA was isolated from sera of 21 patients (14 men, 7 women) before and 3 months after surgery. Sera were combined into two pools, which served for cDNA library construction followed by miSeq sequencing. The levels of candidate miRNAs were validated in the individual samples by QRT-PCR. RESULTS: Serum miR-122 was significantly up-regulated 3 months post-bariatric surgery in sera of patients, whose endothelial function had greatly improved. In addition, serum miR-122 levels correlated positively with endothelial function as measured by FMD. The changes in miR-122 levels from pre-surgery to 3 months post-surgery also tended to correlate with the respective changes in FMD. CONCLUSIONS: The serum miR-122/miR-451 ratio may serve as a marker for endothelial function in obese patients. miR-122 is the dominant miRNA in the liver and a known tumor suppressor. Our findings suggest a role for circulating miR-122 in the maintenance of vascular endothelial cells (VECs) and in the prevention of cancer. Further studies are required to elucidate the mechanism of its secretion into circulation and its absorption by VECs, as well as its relevant cellular targets.

Leptin and insulin up-regulate miR-4443 to suppress NCOA1 and TRAF4, and decrease the invasiveness of human colon cancer cells.

BACKGROUND: Obesity is a risk factor for colorectal cancer (CRC). Normal and tumor cells respond to metabolic hormones, such as leptin and insulin. Thus, obesity-associated resistance to these hormones likely leads to changes in gene expression and behavior of tumor cells. However, the mechanisms affected by leptin and insulin signaling in CRC cells remain mostly unknown. METHODS: We hypothesized that microRNAs (miRNAs) are involved in the regulation of tumorigenesis-related gene expression in CRC cells by leptin and insulin. To test this hypothesis, miRNA levels in the CRC-derived cell lines HCT-116, HT-29 and DLD-1 were profiled, following leptin and insulin treatment. Candidate miRNAs were validated by RT-qPCR. Predicted miRNA targets with known roles in cancer, were validated by immunoblots and reporter assays in HCT-116 cells. Transfection of HCT-116 cells with candidate miRNA mimic was used to test in vitro effects on proliferation and invasion. RESULTS: Of ~800 miRNAs profiled, miR-4443 was consistently up-regulated by leptin and insulin in HCT-116 and HT-29, but not in DLD-1, which lacked normal leptin receptor expression. Dose response experiments showed that leptin at 100 ng/ml consistently up-regulated miR-4443 in HCT-116 cells, concomitantly with a significant decrease in cell invasion ability. Transfection with miR-4443 mimic decreased invasion and proliferation of HCT-116 cells. Moreover, leptin and miR-4443 transfection significantly down-regulated endogenous NCOA1 and TRAF4, both predicted targets of miR-4443 with known roles in cancer metastasis. miR-4443 was found to directly regulate TRAF4 and NCOA1, as validated by a reporter assay. The up-regulation of miR-4443 by leptin or insulin was attenuated by the inhibition of MEK1/2. CONCLUSIONS: Our findings suggest that miR-4443 acts in a tumor-suppressive manner by down-regulating TRAF4 and NCOA1 downstream of MEK-C/EBP-mediated leptin and insulin signaling, and that insulin and/or leptin resistance (e.g. in obesity) may suppress this pathway and increase the risk of metastatic CRC.

Transcriptome variation in banded newt (Ommatotriton vittatus) during its life cycle and habitat transition.

Israel represents the southern limit of the distribution of the banded newt (Ommatotriton vittatus). The life cycle of O. vittatus includes several distinct phases: eggs, aquatic larvae, a terrestrial phase and an aquatic reproductive phase. We investigated differences in gene expression during the life cycle and transition of banded newts between terrestrial and aquatic habitats using mRNA-seq. We identified ∼10 k genes that were differentially expressed (DE) in one of the pairwise comparisons between 3 groups: 1 - terrestrial newts (males and females), 2 - aquatic newts (males and females), 3 - aquatic larvae before metamorphosis. The groups were clearly defined by Principal Components Analysis (PCA). The greatest difference was between aquatic newts (males and females) and aquatic larvae: ∼7.4 k DE genes. Of special interest were the ∼2.4 k genes DE between the aquatic and terrestrial phenotypes. These included prominent candidates with known roles in kidney function (uromodulin homologs were strongly associated with aquatic lifestyle), tissue structure (keratins), and the thyroid hormone signaling modulator DUOXA1. Additional developmental and metabolic pathways overrepresented among the identified DE genes included "epidermis development", "nervous system development", "nucleotide-sugar biosynthesis". Overall, both metamorphosis and environmental transition of banded newts involve extensive transcriptomic remodeling involving developmental, metabolic, and cellular pathways. Understanding the roles of these pathways and individual genes is instrumental for studies of transition between habitats, especially those affected by climate change. Furthermore, the phenotypic flexibility of the newt and the underlying regulation of gene expression can shed light on the evolution of terrestrial vertebrates.

Inversely Regulated Inflammation-Related Processes Mediate Anxiety-Obesity Links in Zebrafish Larvae and Adults.

Anxiety and metabolic impairments are often inter-related, but the underlying mechanisms are unknown. To seek RNAs involved in the anxiety disorder-metabolic disorder link, we subjected zebrafish larvae to caffeine-induced anxiety or high-fat diet (HFD)-induced obesity followed by RNA sequencing and analyses. Notably, differentially expressed (DE) transcripts in these larval models and an adult zebrafish caffeine-induced anxiety model, as well as the transcript profiles of inherently anxious versus less anxious zebrafish strains and high-fat diet-fed versus standard diet-fed adult zebrafish, revealed inversely regulated DE transcripts. In both larval anxiety and obesity models, these included long noncoding RNAs and transfer RNA fragments, with the overrepresented immune system and inflammation pathways, e.g., the "interleukin signaling pathway" and "inflammation mediated by chemokine and cytokine signaling pathway". In adulthood, overrepresented immune system processes included "T cell activation", "leukocyte cell-cell adhesion", and "antigen processing and presentation". Furthermore, unlike adult zebrafish, obesity in larvae was not accompanied by anxiety-like behavior. Together, these results may reflect an antagonistic pleiotropic phenomenon involving a re-adjusted modulation of the anxiety-metabolic links with an occurrence of the acquired immune system. Furthermore, the HFD potential to normalize anxiety-upregulated immune-related genes may reflect the high-fat diet protection of anxiety and neurodegeneration reported by others.

Markers of Genetic Variation in Blue Gourami (Trichogaster trichopterus) as a Model for Labyrinth Fish.

Markers of genetic variation between species are important for both applied and basic research. Here, various genes of the blue gourami (Trichogaster trichopterus, suborder Anabantoidei, a model labyrinth fish), many of them involved in growth and reproduction, are reviewed as markers of genetic variation. The genes encoding the following hormones are described: kisspeptins 1 and 2, gonadotropin-releasing hormones 1, 2, and 3, growth hormone, somatolactin, prolactin, follicle- stimulating hormone and luteinizing hormone, as well as mitochondrial genes encoding cytochrome b and 12S rRNA. Genetic markers in blue gourami, representing the suborder Anabantoidei, differ from those in other bony fishes. The sequence of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene of blue gourami is often used to study the Anabantoidei suborder. Among the genes involved in controlling growth and reproduction, the most suitable genetic markers for distinguishing between species of the Anabantoidei have functions in the hypothalamic-pituitary-somatotropic axis: pituitary adenylate cyclase-activating polypeptide and growth hormone, and the 12S rRNA gene.

Leptin-Responsive MiR-4443 Is a Small Regulatory RNA Independent of the Canonic MicroRNA Biogenesis Pathway.

The human small RNA miR-4443 is functionally involved in several types of cancer and in the biology of the immune system, downstream of insulin and leptin signaling. Next generation sequencing evidence and structural prediction suggest that miR-4443 is not produced via the canonical Drosha-Exportin 5-Dicer pathway of microRNA biogenesis. We tested this hypothesis by using qRT-PCR to measure miR-4443 and other microRNA levels in HCT-116 cells with Drosha, Exportin 5, and Dicer knockouts, as well as in the parental cell line. Neither of the knockouts decreased miR-4443 levels, while the levels of canonical microRNAs (miR-21 and let-7f-5p) were dramatically reduced. Previously published Ago2-RIP-Seq data suggest a limited incorporation of miR-4443 into RISC, in agreement with the functional studies. The miR-4443 locus shows conservation in primates but not in other mammals, while its seed region appears in additional microRNAs. Our results suggest that miR-4443 is a Drosha, Exportin 5, and Dicer-independent, non-canonical small RNA produced by a yet unknown biogenesis pathway.

Serum miR-122 levels correlate with diabetic retinopathy.

Diabetic retinopathy is the most severe ocular complication of diabetes and may lead to visual disability and blindness. Proliferative diabetic retinopathy (PDR) is characterized by ischemia-induced neovascularization with associated complications. An association was established between the presence of PDR, cardiovascular disease, and mortality among patients with type 1 diabetes mellitus and type 2 diabetes mellitus in epidemiological studies. However, the mechanism underlying increased cardiovascular risk in patients with PDR is still unknown. In recent years, a group of miRNAs has been linked to the pathology of diabetes mellitus. Besides, miRNAs in biofluids such as serum have been suggested as potential minimally invasive biomarkers of diabetes and vascular complications. This was a prospective study that recruited 40 human subjects: 10 healthy subjects, 10 with diabetes but without retinopathy (NDR), 10 with diabetic non-proliferative retinopathy (NPDR), and 10 with proliferative diabetic retinopathy (PDR). To examine whether serum miRNAs show altered levels at different stages of diabetic retinopathy, seven specific miRNA candidates (miR-126-3p, miR-130a-3p, miR-21-1, let-7f-5p, miR-122, miR-30c and miR-451a) were measured by qRT-PCR in RNA isolated from sera of all subjects. miR-122 levels increased in parallel with retinopathy severity: from healthy controls to NDR and from NDR to NPDR. However, when the disease progressed to PDR a marked decrease in miR-122 level was noted. This decrease was significant both compared to NPDR samples (p = 0.016) and to all non-PDR samples (p = 0.0002). Additionally, a positive trend was observed comparing miR-122 levels and the number of endothelial progenitor cells in the sera of all subjects. A significant increase in miR-122 was found in patients with diabetic retinopathy that may be related to its role in preventing angiogenesis and proliferation. The dramatic decline in patients with PDR may represent an inhibition or exhaustion of the anti-angiogenic anti-proliferative defense system. Further studies are needed to understand whether miRNA-122 has a role in the pathogenesis of diabetic retinopathy.

Sex-related gonadal gene expression differences in the Russian sturgeon (Acipenser gueldenstaedtii) grown in stable aquaculture conditions.

The Russian sturgeon (Acipenser gueldenstaedtii) is a primitive freshwater fish and a source of caviar. In the present study, the gonadal transcriptomes of male and female Russian sturgeons grown in stable aquaculture conditions were analyzed. RNA sequencing of whole-gonad transcriptomes from pools of 4-year old fish (five females, four males), resulted in the identification of 28,170 unique transcripts. Of these, 16,191 could be annotated by similarity to gene sequences from other species. There were 392 transcripts that showed differential abundance by a factor of 20-fold or more between the sexes; 272 of these were annotated; of these, 175 and 97 were in greater abundance in ovaries and testes, respectively. Functional annotation and clustering of the genes with differential abundances of mRNA allowed for identification of several clusters. Thus, a group of transcriptional regulators and factors involved in cell division, especially septins, were in greater abundance in the ovaries; while a different set of transcription factors (including sox6 and sox30) and a group of protein kinases were in greater abundance in the testes. The transcript abundances of nine highly abundant candidate transcripts, as well as of two additional genes previously known to be involved in reproduction, cyp19 (p450 aromatase) and foxl2, were assessed in the individual samples by qRT-PCR. Of these, five (including cyp19 and foxl2) were in greater abundance in ovaries, while the abundance of ighm1 mRNA was greater in testes. Phylogenetic analysis based on the k1c18 keratin gene placed the sturgeon sequence nearest those of other primitive fish species, supporting the ancient origin of the sturgeon. In conclusion, this study details transcriptome differences between male and female sturgeon and identifies key genes that may contribute to sexual determination and differentiation.

Sex Differences in Plasma MicroRNA Biomarkers of Early and Complicated Diabetes Mellitus in Israeli Arab and Jewish Patients.

MicroRNAs play functional roles in the etiology of type 2 diabetes mellitus (T2DM) and complications, and extracellular microRNAs have attracted interest as potential biomarkers of these conditions. We aimed to identify a set of plasma microRNAs, which could serve as biomarkers of T2DM and complications in a mixed Israeli Arab/Jewish patient sample. Subjects included 30 healthy volunteers, 29 early-stage T2DM patients, and 29 late-stage T2DM patients with renal and/or vascular complications. RNA was isolated from plasma, and the levels of 12 candidate microRNAs were measured by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). MicroRNA levels were compared between the groups and correlated to clinical measurements, followed by stepwise regression analysis and discriminant analysis. Plasma miR-486-3p and miR-423 were respectively up- and down-regulated in T2DM patients compared to healthy controls. MiR-28-3p and miR-423 were up-regulated in patients with complicated T2DM compared to early T2DM, while miR-486-3p was down-regulated. Combined, four microRNAs (miR-146a-5p, miR-16-2-3p, miR-126-5p, and miR-30d) could distinguish early from complicated T2DM with 77% accuracy and 79% sensitivity. In male patients only, the same microRNAs, with the addition of miR-423, could distinguish early from complicated T2DM with 83.3% accuracy. Furthermore, plasma microRNA levels showed significant correlations with clinical measurements, and these differed between men and women. Additionally, miR-183-5p levels differed significantly between the ethnic groups. Our study identified a panel of specific plasma microRNAs which can serve as biomarkers of T2DM and its complications and emphasizes the importance of sex differences in their clinical application.

MicroRNA-423 may regulate diabetic vasculopathy.

To test the hypothesis that microRNAs may play a role in diabetic retinopathy, we measured the levels of different markers [microRNAs, vascular endothelial growth factor (VEGF), nitric oxide (NO), and total antioxidant capacity (TAO)] in patients with type 2 diabetes mellitus (T2DM) and microvascular complications. Sixty-nine patients were recruited: 22 healthy subjects, ten T2DM patients without retinopathy, 22 with nonproliferative diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). Serum levels of NO, VEGF, TAO and 16 candidate microRNAs were measured. Additionally, the mRNA levels of endothelial nitric oxide synthase (eNOS), induced NOS (iNOS), C reactive protein (CRP), VEGF, tumor necrosis factor α (TNFα), PON2, p22, and SOD2 were measured in human vascular endothelial cells cultured in the presence of pooled sera from the subject groups. Plasma miR-423 levels showed a significant ~ twofold decrease in patients with PDR compared to controls. P lasma NO levels were significantly higher in retinopathy, VEGF levels were significantly lower, and TAO was significantly decreased. eNOS mRNA levels were lower in the cells of T2DM patients without retinopathy, but higher in PDR. PON2, p22, and SOD2 mRNA levels were all significantly lower in PDR. CRP, TNFα, iNOS, and VEGF mRNA levels showed no significant association with disease status. Lowered miR-423 levels in diabetic patients showed a correlation with VEGF and an inverse correlation between NO and eNOS expression. Our findings suggest a cross talk between miR-423 and VEGF signaling, affecting eNOS function. miR-423 may be involved in the regulation of diabetic vascular retinal proliferation.

Effects of alkaline-electrolyzed and hydrogen-rich water, in a high-fat-diet nonalcoholic fatty liver disease mouse model.

AIM: To identify the effect of hydrogen-rich water (HRW) and electrolyzed-alkaline water (EAW) on high-fat-induced non-alcoholic fatty acid disease in mice. METHODS: Mice were divided into four groups: (1) Regular diet (RD)/regular water (RW); (2) high-fat diet (HFD)/RW; (3) RD/EAW; and (4) HFD/EAW. Weight and body composition were measured. After twelve weeks, animals were sacrificed, and livers were processed for histology and reverse-transcriptase polymerase chain reaction. A similar experiment was performed using HRW to determine the influence and importance of molecular hydrogen (H2) in EAW. Finally, we compared the response of hepatocytes isolated from mice drinking HRW or RW to palmitate overload. RESULTS: EAW had several properties important to the study: (1) pH = 11; (2) oxidation-reduction potential of -495 mV; and (3) H2 = 0.2 mg/L. However, in contrast to other studies, there were no differences between the groups drinking EAW or RW in either the RD or HFD groups. We hypothesized that the null result was due to low H2 concentrations. Therefore, we evaluated the effects of RW and low and high HRW concentrations (L-HRW = 0.3 mg H2/L and H-HRW = 0.8 mg H2/L, respectively) in mice fed an HFD. Compared to RW and L-HRW, H-HRW resulted in a lower increase in fat mass (46% vs 61%), an increase in lean body mass (42% vs 28%), and a decrease in hepatic lipid accumulation (P < 0.01). Lastly, exposure of hepatocytes isolated from mice drinking H-HRW to palmitate overload demonstrated a protective effect from H2 by reducing hepatocyte lipid accumulation in comparison to mice drinking regular water. CONCLUSION: H2 is the therapeutic agent in electrolyzed-alkaline water and attenuates HFD-induced nonalcoholic fatty liver disease in mice.

MicroRNA modulation of megakaryoblast fate involves cholinergic signaling.

MicroRNAs (miRNAs) are abundant small regulatory RNAs with multiple roles in cell fate determination. The processes regulating cellular miRNA levels are still unclear and experimental oligonucleotide tools to readily mimic their effects are not yet available. Here, we report that thapsigargin-induced intracellular Ca(++) release suppressed pre-miR-181a levels in human promegakaryotic Meg-01 cells, induced differentiation-associated nuclear endoreduplication and caspase-3 activation and replaced the acetylcholinesterase 3' splice variant AChE-S with AChE-R. AChE, PKC and PKA inhibitors all attenuated the pre-miR-181a decline and the induced differentiation. AChmiON, a synthetic 23-mer 2'-oxymethylated oligonucleotide mimicking the miR-181a sequence, blocked the calcium-induced differentiation while elevating cellular pre-miR-181a levels and inducing DNA fragmentation and cell death. Moreover, when added to RW 264.7 macrophages, AChmiON at 100 nM induced nitric oxide production with efficiency close to that of bacterial endotoxin, demonstrating physiologically relevant activities also in blood-born monocytes/macrophages. The stress-induced modulation of hematopoietic miR-181a levels through AChE, PKC and PKA cascade(s) suggests using miRNA mimics for diverting the fate of hematopoietic tumor cells towards differentiation and/or apoptosis.

Prolonged culture of telomerase-immortalized human fibroblasts leads to a premalignant phenotype.

Telomere shortening in primary human fibroblasts results in replicative senescence, which can be overcome by telomerase (hTERT) overexpression. However, because immortalization is one of the hallmarks of malignant transformation, careful analysis of hTERT-immortalized cells is of crucial importance for understanding both processes. To this end, we infected WI-38 fibroblasts with a retrovirus carrying the hTERT cDNA and analyzed their proliferative behavior during 600 days [ approximately 500 population doublings (PDLs)] of continuous culture. Growth of three independent mass cultures was uniform for approximately 150 PDLs after telomerase infection, followed by a progressive acceleration of growth in two of three cultures. Expression of p16(INK4A) was significantly elevated in the immortalized cells but gradually disappeared during the accelerated growth phase. This alteration correlated with loss of the contact inhibition response and conferred the cells with sensitivity to H-Ras-induced transformation. In contrast, the p53- and pRb-mediated checkpoints such as the DNA damage response, chromosomal stability and entry into quiescence remained intact, irrespective of INK4A locus expression. Importantly, detailed examination of one of the WI-38/hTERT cultures during the accelerated growth phase revealed overexpression of the c-myc and Bmi-1 oncogenes, as well as loss of p14(ARF) expression. Collectively, our results indicate that although hTERT-immortalized cells behave similarly to primary cells during the first 150 PDLs, long-term growth in culture may favor the appearance of clones carrying potentially malignant alterations.