CPEB2-dependent translation of long 3'-UTR Ucp1 mRNA promotes thermogenesis in brown adipose tissue.

Expression of mitochondrial proton transporter uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is essential for mammalian thermogenesis. While human UCP1 mRNA exists in a long form only, alternative polyadenylation creates two different isoforms in mice with 10% of UCP1 mRNA found in the long form (Ucp1L) and ~90% in the short form (Ucp1S). We generated a mouse model expressing only Ucp1S and found that it showed impaired thermogenesis due to a 60% drop in UCP1 protein levels, suggesting that Ucp1L is more efficiently translated than Ucp1S. In addition, we found that ß3 adrenergic receptor signaling promoted the translation of mouse Ucp1L and human Ucp1 in a manner dependent on cytoplasmic polyadenylation element binding protein 2 (CPEB2). CPEB2-knockout mice showed reduced UCP1 levels and impaired thermogenesis in BAT, which was rescued by ectopic expression of CPEB2. Hence, long 3'-UTR Ucp1 mRNA translation activated by CPEB2 is likely conserved and important in humans to produce UCP1 for thermogenesis.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

MicroRNA-155 inhibits dengue virus replication by inducing heme oxygenase-1-mediated antiviral interferon responses.

MicroRNAs (miRNAs) have been reported to directly alter the virus life cycle and virus-host interactions, and so are considered promising molecules for controlling virus infection. In the present study, we observed that miR-155 time-dependently downregulated upon dengue virus (DENV) infection. In contrast, exogenous overexpression of miR-155 appeared to limit viral replication in vitro, suggesting that the low levels of miR-155 would be beneficial for DENV replication. In vivo, overexpression of miR-155 protected ICR suckling mice from the life-threatening effects of DENV infection and reduced virus propagation. Further investigation revealed that the anti-DENV activity of miR-155 was due to target Bach1, resulting in the induction of the heme oxygenase-1 (HO-1)-mediated inhibition of DENV NS2B/NS3 protease activity, ultimately leading to induction of antiviral interferon responses, including interferon-induced protein kinase R (PKR), 2'-5'-oligoadenylate synthetase 1 (OAS1), OAS2, and OAS3 expression, against DENV replication. Collectively, our results provide a promising new strategy to manage DENV infection by modulation of miR-155 expression.

ATM and TP53 polymorphisms modified susceptibility to radiation-induced lens opacity in natural high background radiation area, China.

Purpose: A population-based case-control study was conducted in Yangjiang and Enping areas in South China to assess whether the risk of lens opacity induced by natural high background radiation exposure is modulated by polymorphisms of ATM and TP53.Materials and methods: A total of 133 cases who were diagnosed with cortical and posterior subcapsular (PSC) opacity were recruited, and 419 healthy controls were selected through counter-matching in terms of radiation status. Genomic DNA from all the participants was genotyped with the Illumina platform for four single nucleotide polymorphisms of ATM (rs189037, rs373759, and rs4585) and TP53 (rs1042522). The cumulative lens dose received during the entire life was estimated based on annual indoor and outdoor radiation doses and gender- and age-specific occupancy factors. Non-conditional logistic regression was performed to calculate odds ratio (OR) and 95% confidence intervals (95% CI).Results:ATM rs189037 and TP53 rs1042522 were significantly related to cortical and PSC opacity. The risk of opacity was higher when individuals carried the A allele of ATM rs189037 and C allele of TP53 rs1042522, compared with GG genotype. ATM rs189037 A allele carriers (AG/AA) and TP53 rs1042522 C allele carriers (CG/CC) combined with a cumulative lens dose of 100 mGy or higher showed statistically significant opacity risks (OR = 5.51, 95% CI: 1.47-20.66; OR = 2.69, 95% CI: 1.10-6.60).Conclusion: The A allele of ATM rs189037 and C allele of TP53 rs1042522 increased the risk of lens opacity induced by radiation. These polymorphisms in ATM and TP53 might modify the risk of cortical and PSC opacity induced by chronic and prolonged low-dose radiation.

Identification and functional analyses of polymorphism haplotypes of protein phosphatase 2A-Aα gene promoter.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme family that plays an essential regulatory role in cell growth, differentiation, and apoptosis. Mutations in the genes encoding PP2A-Aα/ß subunits are associated with tumorigenesis and other human diseases. To explore whether genetic variations in the promoter region of the PP2A-Aα gene (PPP2R1A) and their frequent haplotypes in the Han Chinese population have an impact on transcriptional activity, we collected DNA samples from 63 healthy Chinese donors and searched for genetic variations in the 5'-flanking promoter region of PPP2R1A (PPP2R1Ap). Haplotypes were characterized by Haploview analysis and individual subcloning. A set of molecular and functional experiments was performed using reporter genes and electrophoretic mobility shifting assay (EMSA). Seven genetic variations were identified within the promoter locus (2038bp) of PPP2R1A. Linkage disequilibrium (LD) patterns and haplotype profiles were analyzed using the identified genetic variants. Using serially truncated human PPP2R1A promoter luciferase constructs, we found that a 685bp (-448nt to +237nt) fragment around the transcription start site (TSS) was the core promoter region. Individual subcloning revealed the existence of six haplotypes in this proximal promoter region of PPP2R1Ap. Using luciferase reporter assays, we found that different haplotypes bearing different variant alleles exhibit distinct promoter activities. The EMSA revealed that the -241 -/G variant influences DNA-protein interactions involving the transcription factor NF-κB, which may regulate the activity of the PPP2R1A proximal promoter. Our findings suggest that functional genetic variants in the proximal promoter of the PP2A-Aα gene and their haplotypes are critical in the regulation of transcriptional activation.

A maternal high-fat diet during pregnancy in rats results in a greater risk of carcinogen-induced mammary tumors in the female offspring than exposure to a high-fat diet in postnatal life.

The association between a high-fat diet and breast cancer risk is controversial. We hypothesized that the exposure of rats to a high-fat diet in utero via the maternal diet would result in a greater risk of carcinogen-induced mammary tumors than high-fat diet exposure in postnatal life. Rats were exposed to a high-fat diet with 40% of the energy source as safflower oil in utero (In utero group), at postnatal days 30-50 (Puberty group), postnatal days 150-170 (Adult group), postnatal days 1-230 (Postnatal group) or for their whole life from in utero (Whole group). Chow diet-fed rats were used as the Nonexposure group. Mammary tumor incidence was significantly higher in the In utero (60%), Postnatal (61%) and Whole (91%) groups than in the Nonexposure group (32%), but there was no significant difference between the Puberty (44%), Adult (44%) and Nonexposure groups. Arachidonic acid levels were 10 times higher in mammary tumor tissue than in the normal mammary gland across all groups and were positively correlated with tumor weight. We conclude that the timing, but not the duration, of high-fat diet exposure makes rats more susceptible to carcinogen-induced mammary tumors and that exposure in utero to a maternal high-fat diet during pregnancy is more important in increasing the risk of mammary tumors in the female offspring than exposure of the offspring to the same high-fat diet later in life.

Three new C-27-carboxylated-lupane-triterpenoid derivatives from Potentilla discolor Bunge and their in vitro antitumor activities.

Three new lupane-triterpenoids (1-3) along with six known compounds (4-9) were isolated from the ethanolic extract of whole plant of Potentilla discolor Bunge. The structures of Compounds 1-3 were established by extensive 1D and 2D NMR together with other spectrum analysis, indicating that their C-27 positions were highly oxygenated, which were rarely found in nature. Their in vitro anti-proliferative activities against HepG-2, MCF-7 and T-84 cell lines were evaluated by Cell Counting Kit-8 (CCK-8) assay, and the results showed different activities for three cell lines with IC50 values ranging from 17.84 to 40.64 µM. In addition, the results from Hoechst 33258 and AO/EB staining as well as annexinV-FITC assays exhibited Compound 1 caused a markedly increased HepG-2 cellular apoptosis in a dose-dependent manner. The further mechanisms of Compound 1-induced cellular apoptosis were confirmed that 1 induced the production of ROS and the alteration of pro- and anti-apoptotic proteins, which led to the dysfunction of mitochondria and activation of caspase-9 and caspase-3 and finally caused cellular apoptosis. These results would be useful in search for new potential antitumor agents and for developing semisynthetic lupane-triterpenoid derivatives with high antitumor activity.

Parthenolide inhibits cancer stem-like side population of nasopharyngeal carcinoma cells via suppression of the NF-κB/COX-2 pathway.

Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.

Exposure to a maternal n-3 fatty acid-deficient diet during brain development provokes excessive hypothalamic-pituitary-adrenal axis responses to stress and behavioral indices of depression and anxiety in male rat offspring later in life.

Brain docosahexaenoic acid (DHA, 22:6n-3) accumulates rapidly during brain development and is essential for normal neurological function. The aim of this study was to evaluate whether brain development was the critical period in which DHA deficiency leads to dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress later in life. Rats were exposed to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet either throughout the preweaning period from embryo to weaning at 3 weeks old or during the postweaning period from 3 to 10 weeks old. Exposure to the n-3 fatty acid-deficient diet during the preweaning period resulted, at weaning, in a significant decrease in hypothalamic DHA levels and a reduced male offspring body weight. DHA deficiency during the preweaning period significantly increased and prolonged restraint stress-induced changes in colonic temperature and serum corticosterone levels, caused a significant increase in GABA(A) antagonist-induced heart rate changes and enhanced depressive-like behavior in the forced swimming test and anxiety-like behavior in the plus-maze test in later life. These effects were not seen in male rats fed the n-3 fatty acid-deficient diet during the postweaning period. These results suggest that brain development is the critical period in which DHA deficiency leads to excessive HPA responses to stress and elevated behavioral indices of depression and anxiety in adulthood. We propose that these effects of hypothalamic DHA deficiency during brain development may involve a GABA(A) receptor-mediated mechanism.

Role of a novel functional variant in the PPP2R1A promoter on the regulation of PP2A-Aalpha and the risk of hepatocellular carcinoma.

Previously, we identified the genetic variant -241 (-/G) (rs11453459) in the PP2A-Aα gene (PPP2R1A) promoter and demonstrated that this variant influences the DNA-binding affinity of nuclear factor-kappa B (NF-κB). In this study, we further confirmed that the transcriptional activity of PPP2R1A may be regulated by NF-κB through the functional genetic variant -241 (-/G). Moreover, we also demonstrated that the methylation status of CpG islands in the promoter of PPP2R1A influences the activity of this gene promoter. Few studies have examined the role of this -241 (-/G) variant in genetic or epigenetic regulation in hepatocellular carcinoma (HCC). To investigate whether this functional variant in the PPP2R1A promoter is associated with the risk of HCC and confirm the function of the -241 (-/G) variant in the HCC population, we conducted a case-control study involving 251 HCC cases and 252 cancer-free controls from a Han population in southern China. Compared with the -241 (--) homozygote, the heterozygous -241 (-G) genotype (adjusted OR  = 0.32, 95% confidence interval (CI)  = 0.17-0.58, P<0.001) and the -241 (-G)/(GG) genotypes (adjusted OR  = 0.38, 95% CI  = 0.22-0.67, P  = 0.001) were both significantly associated with a reduced risk of HCC. Stratification analysis indicated that the protective role of -241 (-G) was more pronounced in individuals who were ≤ 40 years of age, female and HBV-negative. Our data suggest that the transcriptional activity of PPP2R1A is regulated by NF-κB through the -241 (-/G) variant and by the methylation of the promoter region. Moreover, the functional -241 (-/G) variant in the PPP2R1A promoter contributes to the decreased risk of HCC. These findings contribute novel information regarding the gene transcription of PPP2R1A regulated by the polymorphism and methylation in the promoter region through genetic and epigenetic mechanisms in hepatocarcinogenesis.

Fish oil supplementation of maternal rats on an n-3 fatty acid-deficient diet prevents depletion of maternal brain regional docosahexaenoic acid levels and has a postpartum anxiolytic effect.

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are the major polyunsaturated fatty acids (PUFA) in the neuronal membrane. Most DHA and AA accumulation in the brain occurs during the perinatal period via placenta and milk. This study examined whether maternal brain levels of DHA and AA are depleted during pregnancy and lactation due to meeting the high demand of the developing nervous system in the offspring and evaluated the effects of the reproductive cycle on serotonin metabolism and of fish oil (FO) on postpartum anxiety. Pregnant rats were fed during pregnancy and lactation with a sunflower oil-based n-3 PUFA-deficient diet without or with FO supplementation, which provided 0.37% of the energy source as n-3 PUFA, and the age-matched virgin rats were fed the same diets for 41 days. In both sets of postpartum rats, decreased DHA levels compared to those in virgin females were seen in the hypothalamus, hippocampus, frontal cortex, cerebellum, olfactory bulb and retina, while AA depletion was seen only in the hypothalamus, hippocampus and frontal cortex. Serotonin levels were decreased and turnover increased in the brainstem and frontal cortex in postpartum rats compared to virgin rats. FO supplementation during pregnancy and lactation prevented the decrease in maternal brain regional DHA levels, inhibited monoamine oxidase-A activity in the brainstem and decreased anxiety-like behavior. We propose that the reproductive cycle depletes maternal brain DHA levels and modulates maternal brain serotonin metabolism to cause postpartum anxiety and suggest that FO supplementation may be beneficial for postpartum anxiety in women on an n-3 PUFA-deficient diet.

Identification and functional analysis of variant haplotypes in the 5'-flanking region of protein phosphatase 2A-Bδ gene.

Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5'-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5'-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5'-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (-540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5'-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the -462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5'-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.

A maternal high n-6 fat diet with fish oil supplementation during pregnancy and lactation in rats decreases breast cancer risk in the female offspring.

The timing of dietary fat intake may modify breast cancer risk. In addition, n-3 fatty acids reduce, and n-6 fatty acids increase, the risk of breast cancer and a maternal high n-6 fat diet results in a greater risk of breast cancer in the female offspring. We hypothesized that the timing of n-3 fatty acid-enriched fish oil supplementation would be important for reducing the risk of breast cancer. Female rats were fed to a high n-6 fat diet containing 20% of the sunflower oil by weight during pregnancy and lactation, and the female offspring were exposed to fish oil by oral gavage either during the perinatal period via maternal intake or during puberty or adulthood. Exposure during the perinatal period to a maternal high n-6 fat diet with fish oil supplementation significantly reduced the incidence of carcinogen-induced mammary tumors in the female offspring compared to a maternal high n-6 fat diet with no fish oil supplementation or fish oil supplementation later in life (P=.0228 by Cox proportional hazards model). We found that a maternal high n-6 fat diet during pregnancy is more important in increasing the risk of mammary tumors in the female offspring than a maternal high n-6 fat diet during lactation. This study suggests that fish oil supplementation during the perinatal period decreases the effect of a maternal high n-6 fat diet on subsequent carcinogen-induced mammary tumor risk, whereas fish oil supplementation during puberty or adulthood does not.

Contribution of integrons, and SmeABC and SmeDEF efflux pumps to multidrug resistance in clinical isolates of Stenotrophomonas maltophilia.

OBJECTIVES: The contribution of integrons and efflux pumps to multidrug resistance in Stenotrophomonas maltophilia was evaluated. MATERIALS AND METHODS: Ninety-three S. maltophilia clinical isolates were studied. PCR and direct sequencing were used to detect the presence of integrons. Real-time PCR was performed to assess and quantify the expression of the Sme efflux pumps of S. maltophilia. RESULTS: Class 1 integrons were detected in 22% of clinical isolates and carried cassettes conferring resistance mainly to aminoglycosides and trimethoprim. The small multidrug resistance gene, smr, was found on class 1 integrons in six isolates. Thirty-one percent of the isolates overexpressed the smeDEF gene, as compared with a control strain, and 59% overexpressed the smeABC gene. Extrusion of ciprofloxacin and meropenem was specific to the SmeABC and SmeDEF pumps, respectively. CONCLUSION: SmeABC and SmeDEF efflux pumps play important roles in resistance of S. maltophilia to ciprofloxacin and meropenem.