PTEN and rapamycin inhibiting the growth of K562 cells through regulating mTOR signaling pathway

<p>Abstract</p> <p>Objective</p> <p>To investigate, <it>in vitro</it>, the regulatory effects of tumor-suppressing gene PTEN on mTOR (mammalian target of rapamycin) signaling pathway, the effects of transfected PTEN and rapamycin on the growth inhibition, and apoptosis induction for human leukemia cell line K562 cells.</p> <p>Methods</p> <p>K562 cells were transfected with recombined adenovirus-PTEN vector containing green fluorescent protein (Ad-PTEN-GFP), followed by the treatment of the cells with or without rapamycin. The proliferation inhibition rate and apoptotic rate of these transfected and/or rapamycin treated K562 cells were measured by MTT assay and flow cytometry (FCM), the expression levels of PTEN-, mTOR-, cyclinD1- and P27^kip1- mRNA were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR), the protein expression levels of PTEN, Akt, p-Akt were detected by western blotting.</p> <p>Results</p> <p>The proliferation of K562 cells was inhibited by PTEN gene transfection with/without the treatment of rapamycin. The expression levels of PTEN- and P27^kip1- mRNA were up-regulated, and the mTOR- and cyclinD1- mRNA were down-regulated in K562 cells after the cells transfected with wild type PTEN gene and treated with rapamycin.</p> <p>Conclusion</p> <p>PTEN and rapamycin inhibited mTOR expression by acting as an upstream regulator of mTOR. Low dose rapamycin in combination with over-expressed PTEN might have synergistic effects on inhibiting the proliferation and promoting apoptosis of K562 cells.</p

The depth-profiled carrier concentration and scattering mechanism in undoped GaN film grown on sapphire

The carrier concentration and scattering mechanism in undoped GaN film grown on sapphire were investigated. The film was grown on sapphire using metal organic chemical vapor deposition (MOCVD). Confocal micro-Raman spectroscopic measurements and temperature-dependant Hall (TDH) measurements were performed for the study of the depth distribution of the carrier density across the GaN film. The existence of a nonuniform spatial distribution of free carriers in the film with a highly conductive layer of ∼1 μm thickness near the GaN sapphire boundary was confirmed from the study. The electron mobility limiting effect of nitrogen vacancies on GaN bulk film was also discussed.published_or_final_versio

Chromosome‑level assembly and analysis of \u3ci\u3eCamelina neglecta\u3c/i\u3e: a novel diploid model for Camelina biotechnology research

Camelina neglecta is a new diploid Brassicaceae species, which has great research value because of its close relationship with the hexaploid oilseed crop Camelina sativa. Here, we report a chromosome-level assembly of C. neglecta with a total length of 210 Mb. By adopting PacBio sequencing and Hi-C technology, the C. neglecta genome was assembled into 6 chromosomes with scaffold N50 of 29.62 Mb. C. neglecta has undergone the whole-genome triplication (γ) shared among eudicots and two whole-genome duplications (α and β) shared by crucifers, but it has not undergone a specific whole-genome duplication event. By synteny analysis between C. neglecta and C. sativa, we successfully used the method of calculating Ks to distinguish the three subgenomes of C. sativa and determined that C. neglecta was closest to the first subgenome (SG1) of C. sativa. Further, transcriptomic analysis revealed the key genes associated with seed oil biosynthesis and its transcriptional regulation, including SAD, FAD2, FAD3, FAE1, ABI3, WRI1 and FUS3 displaying high expression levels in C. neglecta seeds. The high representability of C. neglecta as a model species for Camelina-based biotechnology research has been demonstrated for the first time. In particular, floral Agrobacterium tumefaciens infiltration-based transformation of C. neglecta, leading to overexpression of CvLPAT2, CpDGAT1 and CvFatB1 transgenes, was demonstrated for medium-chain fatty acid accumulation in C. neglecta seed oil. This study provides an important genomic resource and establishes C. neglecta as a new model for oilseed biotechnology research

Genetic Vaccination-Induced Immune Responses to the Human Immunodeficiency Virus Protein Rev: Emergence of the Interleukin 2-Producing Helper T Lymphocyte

Overview summary The immune system poses a major obstacle to the long-term success of in vivo gene therapies. Immune responses to foreign transgene products and/or the vectors that facilitate gene transfer may neutralize the transgene product, eliminate transfected cells, and culminate in inflammation within transfected tissues. The majority of studies that address these issues have focused on cytotoxic T lymphocyte (CTL) and antibody responses induced by gene transfer. However, the IL-2-producing helper T lymphocyte (HTL) represents a critical regulatory cell that likely influences the inductive phase of the immune response following gene transfer. The current study employed limiting dilution analysis (LDA) techniques to characterize the development of IL-2-producing HTLs induced by genetic vaccination with a plasmid encoding the mutated HIV protein Rev M10. Further, we assessed the ability to inhibit the transgene-induced HTL response by cotransfer of a plasmid encoding the immunosuppressive cytokine TGFβ1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63282/1/hum.1998.9.15-2187.pd

Expansion of Myeloid-Derived Suppressor Cells Promotes Differentiation of Regulatory T Cells in HIV-1+ Individuals

Objective: Regulatory T cells (Tregs) contribute to HIV-1 disease progression by impairing antiviral immunity; however, the precise mechanisms responsible for the development of Tregs in the setting of HIV-1 infection are incompletely understood. Design: In this study, we provide evidence that HIV-induced expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) promote the differentiation of Foxp3+ Tregs. Methods: We measured MDSC induction and cytokine expression by flow cytometry and analyzed their functions by coculturing experiments. Results: We observed a dramatic increase in M-MDSC frequencies in the peripheral blood of HIV-1 seropositive (HIV-1+) individuals, even in those on antiretroviral therapy with undetectable viremia, when compared with healthy participants. We also observed increases in M-MDSCs after incubating healthy peripheral mononuclear cells (PBMCs) with HIV-1 proteins (gp120 or Tat) or Toll-like receptor 4 ligand lipopolysaccharides in vitro, an effect that could be abrogated in the presence of the phosphorylated signal transducer and activator of transcription 3 inhibitor, STA-21. Functional analyses indicated that M-MDSCs from HIV-1+ individuals express higher levels of IL-10, tumor growth factor-β, IL-4 receptor α, p47phex, programmed death-ligand 1, and phosphorylated signal transducer and activator of transcription 3 – all of which are known mediators of myelopoiesis and immunosuppression. Importantly, incubation of healthy CD4+ T cells with MDSCs derived from HIV-1+ individuals significantly increased differentiation of Foxp3+ Tregs. In addition, depletion of MDSCs from PBMCs of HIV-1+ individuals led to a significant reduction of Foxp3+ Tregs and increase of IFNγ production by CD4+ T effector cells. Conclusions: These results suggest that HIV-induced MDSCs promote Treg cell development and inhibit T cell function – a hallmark of many chronic infectious diseases

Arsenic and Fluoride Exposure in Drinking Water: Children’s IQ and Growth in Shanyin County, Shanxi Province, China

BACKGROUND: Recently, in a cross-sectional study of 201 children in Araihazar, Bangladesh, exposure to arsenic (As) in drinking water has been shown to lower the scores on tests that measure children’s intellectual function before and after adjustment for sociodemographic features. OBJECTIVES: We investigated the effects of As and fluoride exposure on children’s intelligence and growth. METHODS: We report the results of a study of 720 children between 8 and 12 years of age in rural villages in Shanyin county, Shanxi province, China. The children were exposed to As at concentrations of 142 ± 106 μg/L (medium-As group) and 190 ± 183 μg/L (high-As group) in drinking water compared with the control group that was exposed to low concentrations of As (2 ± 3 μg/L) and low concentrations of fluoride (0.5 ± 0.2 mg/L). A study group of children exposed to high concentrations of fluoride (8.3 ± 1.9 mg/L) but low concentrations of As (3 ± 3 μg/L) was also included because of the common occurrence of elevated concentrations of fluoride in groundwater in our study area. A standardized IQ (intelligence quotient) test was modified for children in rural China and was based on the classic Raven’s test used to determine the effects of these exposures on children’s intelligence. A standardized measurement procedure for weight, height, chest circumference, and lung capacity was used to determine the effects of these exposures on children’s growth. RESULTS: The mean IQ scores decreased from 105 ± 15 for the control group, to 101 ± 16 for the medium-As group (p < 0.05), and to 95 ± 17 for the high-As group (p < 0.01). The mean IQ score for the high-fluoride group was 101 ± 16 and significantly different from that of the control group (p < 0.05). Children in the control group were taller than those in the high-fluoride group (p < 0.05); weighed more than the those in the high-As group (p < 0.05); and had higher lung capacity than those in the medium-As group (p < 0.05). CONCLUSIONS: Children’s intelligence and growth can be affected by high concentrations of As or fluoride. The IQ scores of the children in the high-As group were the lowest among the four groups we investigated. It is more significant that high concentrations of As affect children’s intelligence. It indicates that arsenic exposure can affect children’s intelligence and growth

HCV-induced miR146a Controls SOCS1/STAT3 and Cytokine Expression in Monocytes to Promote Regulatory T-cell Development

Host innate and adaptive immune responses must be tightly regulated by an intricate balance between positive and negative signals to ensure their appropriate onset and termination while fighting pathogens and avoiding autoimmunity; persistent pathogens may usurp these regulatory machineries to dampen host immune responses for their persistence in vivo. Here, we demonstrate that miR146a is up‐regulated in monocytes from hepatitis C virus (HCV )‐infected individuals compared to control subjects. Interestingly, miR146a expression in monocytes without HCV infection increased, whereas its level in monocytes with HCV infection decreased, following Toll‐like receptor (TLR ) stimulation. This miR146a induction by HCV infection and differential response to TLR stimulation were recapitulated in vitro in monocytes co‐cultured with hepatocytes with or without HCV infection. Importantly, inhibition of miR146a in monocytes from HCV ‐infected patients led to a decrease in IL ‐23, IL ‐10 and TGF ‐β expressions through the induction of suppressor of cytokine signalling 1 (SOCS 1) and the inhibition of signal transducer and activator transcription 3 (STAT 3), and this subsequently resulted in a decrease in regulatory T cells (Tregs) accumulated during HCV infection. These results suggest that miR146a may regulate SOCS 1/STAT 3 and cytokine signalling in monocytes, directing T‐cell differentiation and balancing immune clearance and immune injury during chronic viral infection

Selective Oxidative Stress Induces Dual Damage to Telomeres and Mitochondria in Human T Cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress-mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1O2) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X-ray repair cross-complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging-associated diseases

Inhibition of TRF2 Accelerates Telomere Attrition and DNA Damage in Naïve CD4 T Cells During HCV Infection

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases

Trace the Accretion Geometry of H 1743--322 with Type C Quasi-periodic Oscillations in Multiple Outbursts

We present a systematic analysis of type C quasi-periodic oscillation (QPO) observations of H 1743--322 throughout the Rossi X-ray Timing Explorer (RXTE) era. We find that, while different outbursts have significant flux differences, they show consistent positive correlations between the QPO fractional root-mean-square (rms) amplitude and non-thermal fraction of the emission, which indicate an independence of the intrinsic QPO rms on individual outburst brightness in H 1743--322. However, the dependence of the QPO rms on frequency is different between the outburst rise and decay phases, where QPO fractional rms of the decay phase is significantly lower than that of the rise phase at low frequencies. The spectral analysis also reveals different ranges of coronal temperature between the two outburst stages. A semi-quantitative analysis shows that the Lense-Thirring precession model could be responsible for the QPO rms differences, requiring a variable coronal geometric shape. However, the variable-Comptonization model could also account for the findings. The fact that the rms differences and the hysteresis traces in the hardness-intensity diagram (HID) accompany each other indicates a connection between the two phenomena. By correlating the findings with QPO phase lags and the quasi-simultaneous radio flux previously published, we propose there could be corona-jet transitions in H 1743--322 similar to those that have been recently reported in GRS 1915+105.Comment: 21 pages, 12 figure