High-dimensional Inference for Generalized Linear Models with Hidden Confounding

Statistical inferences for high-dimensional regression models have been extensively studied for their wide applications ranging from genomics, neuroscience, to economics. In practice, there are often potential unmeasured confounders associated with both the response and covariates, leading to the invalidity of the standard debiasing methods. This paper focuses on a generalized linear regression framework with hidden confounding and proposes a debiasing approach to address this high-dimensional problem by adjusting for effects induced by the unmeasured confounders. We establish consistency and asymptotic normality for the proposed debiased estimator. The finite sample performance of the proposed method is demonstrated via extensive numerical studies and an application to a genetic dataset

Evaluating the relationship between Clinical G6PD enzyme activity and gene variants

Glucose-6-phosphate dehydrogenase (G6PD) is a the first and rate-limiting enzyme that plays a critical role in G6PD deficiency, the most common enzyme disorder worldwide, is related to intravascular hemolysis. To determine the clinical enzyme activity level in different G6PD variants, we evaluated 15 variant from 424 clinical blood samples by using multicolor melting curve analysis and DNA sequencing. The results showed that the enzyme activities of the hemizygous deficient were 1.5–2.4 U/gHb, which was significantly lower than those of the heterozygous (P T (Chinese-5) mutation affects the kinetic parameters of G6PD and increase utilization of analogues, its enzyme activity is more than those of other mutations that mutated in the β+α region of G6PD. The heterozygous enzyme levels ranged from 6.5–20.1 U/gHb; and there was no significant difference among different heterozygous variants (P > 0.05). The enzyme activity levels of the compound heterozygous mutation were mainly in the range of 1.7–3.8 U/gHb, which was much lower than that of the heterozygous mutation (P < 0.001). In summary, our findings revealed that the enzyme activity of G6PD in blood have a significant relationship with genotype of G6PD

MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitin-degradation to promote colorectal cancer malignance

Abstract Background MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, high expresses in various cancers and is involved in cancer pathogenesis. However, mechanisms underlying its regulation in carcinogenesis are unclear. Methods The tissue microarray of colorectal cancer (CRC) was used to evaluate MNAT1 expressions in CRC tissues using immunohistochemistry, CRC cell lines were also detected MNAT1 expression using Western-blotting. MNAT1 and shMNAT1 vectors were constructed, and transfected into CRC cells. Cell growths of the transfected cells were observed using MTT and colony formation. The affects of MNAT1 on p53 expression were analyzed using Western-blotting and Real-time PCR. Immunoprecipitation assay was used to analyze the interaction p53 and MNAT1, and Western-blotting was used to test the effects of MNAT1 on p53 downstream molecules. The apoptosis of CRC cells with MNAT1 or shMNAT1 were analyzed using flow cytometry. BABL/c athymic nude mice were used to observe the effect of MNAT1 on CRC cell growth in vivo. Results MNAT1 was found to be overexpressed in CRC tissues and cells, and MNAT1 expressions in CRC tissue samples were associated with CRC carcinogenesis and poor patient outcomes. MNAT1-knockin increased CRC cell growth and colony formation, and MNAT1-knockdown dramatically decreased cell motility and invasion. MNAT1 physically interacted with p53, MNAT1 also increased the interaction of MDM2 with p53. Strikingly, MNAT1 mediated p53 ubiquitin-degradation. MNAT1 shortened p53 half-life, and ectopic MNAT1 expression decreased p53 protein stability. Moreover, MNAT1 induced RAD51 and reduced p21, cleaved-caspase3, cleaved-PARP and BAX expression. MNAT1 inhibited CRC cell apoptosis. shMANT1 decreased tumor growths in nude mice following p53 increase. Conclusion MNAT1 binds to p53, mediates p53 ubiquitin-degradation through MDM2, increases cell growth and decreases cell apoptosis, and finally promotes CRC malignance. MNAT1 binding to p53 and mediating p53 ubiquitin-degradation axis represents a novel molecular joint in the p53 pathway

Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis

N,N\u27-dinitrosopiperazine (DNP) with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC) metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B) motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selected phosphoproteins were verified by Western-blotting analysis. DNP treatment altered the phosphorylation of ezrin (threonine 567), vimentin (serine 55), stathmin (serine 25) and STAT3 (serine 727). Furthermore, it was shown that DNP-dependent metastasis is mediated in part through ezrin at threonine 567, as DNP-mediated metastasis was decreased when threonine 567 of ezrin was mutated. Strikingly, NPC metastatic tumors exhibited a higher expression of phosphorylated-ezrin at threonine 567 than the primary tumors. These findings provide novel insight into DNP-induced NPC metastasis and may contribute to a better understanding of the metastatic mechanisms of NPC tumors

An Aptamer-Array-Based Sample-to-Answer Biosensor for Ochratoxin A Detection via Fluorescence Resonance Energy Transfer

Food toxins are a hidden threat that can cause cancer and tremendously impact human health. Therefore, the detection of food toxins in a timely manner with high sensitivity is of paramount importance for public health and food safety. However, the current detection methods are relatively time-consuming and not practical for field tests. In the present work, we developed a novel aptamer-chip-based sample-to-answer biosensor (ACSB) for ochratoxin A (OTA) detection via fluorescence resonance energy transfer (FRET). In this system, a cyanine 3 (Cy3)-labeled OTA-specific biotinylated aptamer was immobilized on an epoxy-coated chip via streptavidin-biotin binding. A complementary DNA strand to OTA aptamer at the 3′-end was labeled with a black hole quencher 2 (BHQ2) to quench Cy3 fluorescence when in proximity. In the presence of OTA, the Cy3-labeled OTA aptamer bound specifically to OTA and led to the physical separation of Cy3 and BHQ2, which resulted in an increase of fluorescence signal. The limit of detection (LOD) of this ACSB for OTA was 0.005 ng/mL with a linearity range of 0.01–10 ng/mL. The cross-reactivity of ACSB against other mycotoxins, ochratoxin B (OTB), aflatoxin B1 (AFB1), zearalenone (ZEA), or deoxynilvalenol (DON), was less than 0.01%. In addition, this system could accurately detect OTA in rice samples spiked with OTA, and the mean recovery rate of the spiked-in OTA reached 91%, with a coefficient of variation (CV) of 8.57–9.89%. Collectively, the ACSB may represent a rapid, accurate, and easy-to-use platform for OTA detection with high sensitivity and specificity

Immunohistochemical staining of AGR2, CTSB, and CTSD expressions in normal nasopharyngeal epithelial tissues (NNET), nasopharyngeal carcinoma (NPC), and cervical lymph node metastatic NPC (LMNPC) tissues.

<p>The formalin-fixed and paraffin-embedded NNET, NPC, and LMNPC tissue sections were stained using a standard immunohistochemical technique. a, AGR2 expression in NNET; b, AGR2 expression in NPC; c AGR2 expression in LMNPC; d, CTSB expression in NNET; e, CTSB expression in NPC; f, CTSB expression in LMNPC; g, CTSD expression in NNET; h, CTSD expression in NPC; i, CTSD expression in LMNPC; j, NNET hematoxylin and eosin; k, NPC hematoxylin and eosin; l, LMNPC hematoxylin and eosin. Arrows, positive cells. Original magnification, ×400. Scale bar, 5 μm.</p

Differential AGR2, CTSB, and CTSD expression among NNET, NPC, and LMNPC tissues.

<p>Note: *, NNET versus NPC, **, NPC versus LMNPC, ***, LMNPC versus NNET.</p

si-AGR2 decreased DNP-mediated invasion and motility in 5–8F cells.

<p>5–8F cells were transfected with pUprosi-AGR2 (si-AGR2) or pUprosi-mock (si-mock), and 5–8F-si-AGR2 and 5–8F-si-mock were obtained by selection for G418 resistance, and treated with DNP. A, AGR2 expression was detected using western blotting. A Boyden chamber was used to measure the invasion and motility of 5–8F-si-AGR2 and 5–8F-si-mock with or without DNP treatment. The cells that invaded the membrane were fixed with methanol and stained with hematoxylin and eosin. Random fields of view were counted to determine the number of invading cells. B, invasion of the treated cells. C, motility of the treated cells. The number of traversed cells was counted in three individual experiments and presented as the mean ± SD. *indicates P<0.05.</p

DNP-mediated invasion and motility through AGR2 in 6–10B cells.

<p>6–10B cells were transiently transfected with siRNA-AGR2 or si-mock, and then treated with DNP. A, AGR2 expression was detected using western blotting. A Matrigel-coated Boyden chamber was used to measure 6–10B cell invasion, and an uncoated Boyden chamber was used to determine cell motility. The cells that invaded the membrane were fixed with methanol and stained with hematoxylin and eosin. Images were taken under a light microscope and random fields of view were counted to determine the number of invading cells. B, cell invasion of the treated cells. a, 6–10B cells transfected with si-mock and treated with 0.1% DMSO; b, 6–10B cells transfected with si-mock and treated with 8 μM DNP; c, the transfect with siRNA-AGR2 plus DMSO treatment; d, the transfect with siRNA-AGR2 and treated with DNP. C, cell motility of the treated cells. a, 6–10B cells transfected with si-mock and treated with DMSO; b, 6–10B cells transfected with si-mock and treated with DNP; c, the transfect with siRNA-AGR2 and treated with DMSO; d, the transfect with siRNA-AGR2 and treated with DNP. D, invasive cells were counted. E, motile cells were counted. The number of traversed cells were counted in three individual experiments and presented as the mean ± SD. *indicates <i>P</i><0.05.</p

AGR2 interacts with CTSB/CTSD.

<p>A, interaction of endogenous AGR2 and CTS in 6–10B cells. Endogenous AGR2 and CTS expression in 6–10B cells was detected using an immunofluorescence assay. a, AGR2 expression in 6–10B cells; b, CTS expression in 6–10B cells; c, merge of a and b; d, DAPI staining. B, 6–10B cells were respectively transfected with siRNA-AGR2 or si-mock, and then treated with DNP. AGR2 was immunoprecipitated using AGR2 antibody, and CTSB and CTSD were detected in the immunoprecipitates using western blotting. IgG served as the loading control. *indicates <i>P</i><0.05. One representative experiment is presented.</p