Downregulation of PHLPP Expression Contributes to Hypoxia-Induced Resistance to Chemotherapy in Colon Cancer Cells

Hypoxia is a feature of solid tumors. Most tumors are at least partially hypoxic. This hypoxic environment plays a critical role in promoting resistance to anticancer drugs. PHLPP, a novel family of Ser/Thr protein phosphatases, functions as a tumor suppressor in colon cancers. Here, we show that the expression of both PHLPP isoforms is negatively regulated by hypoxia/anoxia in colon cancer cells. Interestingly, a hypoxia-induced decrease of PHLPP expression is attenuated by knocking down HIF1α but not HIF2α. Whereas the mRNA levels of PHLPP are not significantly altered by oxygen deprivation, the reduction of PHLPP expression is caused by decreased protein translation downstream of mTOR and increased degradation. Specifically, hypoxia-induced downregulation of PHLPP is partially rescued in TSC2 or 4E-BP1 knockdown cells as the result of elevated mTOR activity and protein synthesis. Moreover, oxygen deprivation destabilizes PHLPP protein by decreasing the expression of USP46, a deubiquitinase of PHLPP. Functionally, downregulation of PHLPP contributes to hypoxia-induced chemoresistance in colon cancer cells. Taken together, we have identified hypoxia as a novel mechanism by which PHLPP is downregulated in colon cancer, and the expression of PHLPP may serve as a biomarker for better understanding of chemoresistance in cancer treatment.     

CircGFRA1 facilitates the malignant progression of HER-2-positive breast cancer via acting as a sponge of miR-1228 and enhancing AIFM2 expression

CircRNAs (circular RNA) are reported to regulate onset and progress multiple cancers. Nonetheless, the function along with the underlying mechanisms of circRNAs in HER-2-positive breast cancer (BC) remains unclear. CircRNA microarrays were performed to elucidate expression profiles of HER-2-positive BC cells. circRNA levels were quantified using qRT-PCR assay. Various in vitro along with in vivo assays were employed to further explore the effects of circGFRA1 in the progress of HER-2-positive BC and interactions of circGFRA1, miR-1228 and AIFM2 in Her-2-positive BC. CircGFRA1 was remarkably upregulated in HER-2-positive BC. Knockdown of circGFRA1 could attenuate HER-2-positive BC progression by inhibiting the proliferation, infiltration and migratory ability of HER-2-positive BC cells. Through ceRNA mechanism, circGFRA1 could bind to miR-1228 and alleviate inhibitory activity of miR-1228 on targeted gene AIFM2. In summary, circGFRA1-miR-1228-AIFM2 axis regulates HER-2-positive BC. CircGFRA1 is a novel promising treatment option for HER-2-positive BC.     

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3^∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3^∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3^∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.     

An Imputation Approach for Oligonucleotide Microarrays

Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as “bright spots”, “dark clouds”, and “shadowy circles”, etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.     

Noninvasive and Targeted Gene Delivery into the Brain Using Microbubble-Facilitated Focused Ultrasound

Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×10⁹ vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.     

Expression of SFRP Family Proteins in Human Keratoconus Corneas

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.     

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

1. Download : Download high-res image (197KB)
2. Download : Download full-size image

     

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca²⁺] _i mobilization. In the present study, hypoxia exposure of rat's model was established. Two-pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh-TPC1 or Lsh-TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP-activated [Ca²⁺]_i shows to be mediated via two-pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia-induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh-TPC1 or Lsh-TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP-AM-induced PASMC proliferation and [Ca²⁺]_i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP-AM- induced [Ca²⁺]_i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.     

Crude Oil Treatment Leads to Shift of Bacterial Communities in Soils from the Deep Active Layer and Upper Permafrost along the China-Russia Crude Oil Pipeline Route

The buried China-Russia Crude Oil Pipeline (CRCOP) across the permafrost-associated cold ecosystem in northeastern China carries a risk of contamination to the deep active layers and upper permafrost in case of accidental rupture of the embedded pipeline or migration of oil spills. As many soil microbes are capable of degrading petroleum, knowledge about the intrinsic degraders and the microbial dynamics in the deep subsurface could extend our understanding of the application of in-situ bioremediation. In this study, an experiment was conducted to investigate the bacterial communities in response to simulated contamination to deep soil samples by using 454 pyrosequencing amplicons. The result showed that bacterial diversity was reduced after 8-weeks contamination. A shift in bacterial community composition was apparent in crude oil-amended soils with Proteobacteria (esp. α-subdivision) being the dominant phylum, together with Actinobacteria and Firmicutes. The contamination led to enrichment of indigenous bacterial taxa like Novosphingobium, Sphingobium, Caulobacter, Phenylobacterium, Alicylobacillus and Arthrobacter, which are generally capable of degrading polycyclic aromatic hydrocarbons (PAHs). The community shift highlighted the resilience of PAH degraders and their potential for in-situ degradation of crude oil under favorable conditions in the deep soils.