Knockdown of the Ufm1 system resulted in activation of UPR.

<p><b>A.</b> Up-regulation of ER chaperone proteins and CHOP in U2OS cells with knockdown of the Ufm1 system. U2OS cells were infected with lentiviral shRNAs, selected with puromycin (1.5 µg/ml). Cell lysates were collected after 6-day incubation and subject to immunoblotting with indicated antibodies. Knockdown of the Ufm1 components were confirmed by immunoblotting. <b>B.</b> Immunostaining of PDI in Uba5 and Ufc1 knockdown cells. C. Immunostaining of PDI in C53/LZAP and RCAD/Ufl1 knockdown cells. U2OS cells were subjected to immunostaining of PDI,. Knockdown of Uba5 was confirmed by Uba5 staining, while knockdown of RCAD/Ufl1 and C53/LZAP was confirmed by C53 staining. The images were acquired by Zeiss Axio Observer D1 and Axiovision software.</p

Expression of the Ufm1 system was induced by ER stress.

<p><b>A.</b> RT-PCR results of HCT116 and HepG2 cells treated with TG (1 µM for 16 hours) and TM (10 µM for 16 hours). The results represented mean ± SEM, p value <0.01 (marked by *). The inserts in Fig. 2A showed the results of Xbp-1 splicing assays. “u” indicated the unspliced form of Xbp-1, while “s” is the spliced form. <b>B.</b> Immunoblotting of the cell lysates of HCT116 and HepG2 treated with TG and TM. Specific Ufm1 conjugates were marked by arrowheads, and the non-specific 70 kD band was indicated by a star. “Long exp.” was long exposure of the blot, while “Short exp.” was short exposure of the blot in Enhanced Chemiluminescence (ECL).</p

Transcriptional Regulation of the Ufm1 Conjugation System in Response to Disturbance of the Endoplasmic Reticulum Homeostasis and Inhibition of Vesicle Trafficking

<div><p>Homeostasis of the endoplasmic reticulum (ER) is essential for normal cellular functions. Disturbance of this homeostasis causes ER stress and activates the Unfolded Protein Response (UPR). The Ufm1 conjugation system is a novel Ubiquitin-like (Ubl) system whose physiological target(s) and biological functions remain largely undefined. Genetic study has demonstrated that the Ufm1-activating enzyme Uba5 is indispensible for erythroid differentiation in mice, highlighting the importance of this novel system in animal development. In this report we present the evidence for involvement of RCAD/Ufl1, a putative Ufm1-specific E3 ligase, and its binding partner C53/LZAP protein in ufmylation of endogenous Ufm1 targets. Moreover, we found that the Ufm1 system was transcriptionally up-regulated by disturbance of the ER homeostasis and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, we dissected the Ufm1 promoter and found that Ufm1 was a potential target of Xbp-1, one of crucial transcription factors in UPR. We further examined the effect of Xbp-1 deficiency on the expression of the Ufm1 components. Interestingly, the expression of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type mouse embryonic fibroblasts (MEFs) was significantly induced by inhibition of vesicle trafficking, but the induction was negated by Xbp-1 deficiency. Finally, we found that knockdown of the Ufm1 system in U2OS cells triggered UPR and amplification of the ER network. Taken together, our study provided critical insight into the regulatory mechanism of the Ufm1 system and established a direct link between this novel Ubl system and the ER network.</p> </div

The effect of the UPR pathways on the expression of the Ufm1 system.

<p><b>A.</b> Immunoblotting of MEF cell lysates using Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP antibodies. GAPDH was used as a loading control. Relative ratios of proteins were measured against GAPDH using Image J software. <b>B.</b> The mRNA levels of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type and Xbp-1^−/− MEF cells that were treated with ER stress-inducing agents (TG, 0.5 µM for 16 hour; TM, 10 µM for 16 hours; and BFA, 0.5 µg/ml for 16 hours). <b>C.</b> The mRNA levels of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type and PERK^−/− MEF cells that were treated with ER stress-inducing agents. The results represented mean ± SD. *p value <0.01.</p

Ufm1 is a potential target of Xbp-1.

<p><b>A.</b> The constructs of the Ufm1 promoter used for luciferase reporter assays. Human Ufm1 promoter sequence was amplified from the genome of HCT116 cells. <b>B.</b> The minimal Ufm1 promoter responded to ER stresses. Various Ufm1 promoter constructs were transfected into 293T cells that were subsequently treated with TG (0.5 µM) and TM (10 µM) for 24 hours. The promoter activity was measured by dual luciferase reporter assays (Promega). <b>C.</b> The putative Xbp-1 binding site was responsible for Xbp-1-mediated induction of Ufm1. 293T cells were transfected with indicated Ufm1 promoter constructs along with Xbp-1 expression vector. The cells were subsequently treated with TG and TM, and the promoter activity was measured by dual luciferase assays. <b>D.</b> The Ufm1 promoter activity in wild-type and Xbp-1^−/− MEFs. The Ufm1 protomer reporter was transfected into wild-type and Xbp-1^−/− MEFs, and the promoter activity was measured by dual luciferase reporter assays (Promega). The results represented mean ± SD. *p value <0.01 and **p value <0.05. <b>E.</b> CHIP assay. The Xbp-1-DNA complex was immunoprecipitated with Xbp-1 antibody, and subject to PCR using the primers specific for Ufm1 and ERdj4 promoters.</p

Coaxial technique-promoted diagnostic accuracy of CT-guided percutaneous cutting needle biopsy for small and deep lung lesions

<div><p>Coaxial technique is extensively applied to facilitate percutaneous lung lesion biopsy. However, the impact of coaxial technique on diagnostic accuracy remains undecided. We reviewed 485 patients who underwent percutaneous CT-guided needle biopsies of lung lesions in our hospital. All of these biopsies were performed using either a cutting needle alone (n = 268) or a cutting needle combined with a coaxial needle (n = 217). The diagnostic accuracy and complications resulting from the two techniques were then compared. The diagnostic accuracies of the two techniques were comparably high, at 98.2% (with coaxial technique) and 95.9% (without coaxial technique), <i>p</i> = 0.24. Subgroup analysis discovered that for patients with lesions measuring < 1.5 cm and needle path length ≥ 4 cm, the coaxial technique achieved a higher diagnostic accuracy (95.5% vs. 72.7%, <i>p</i> = 0.023). The biopsy was well tolerated in all of the patients. Pneumothorax occurred less often in patients who were biopsied with the coaxial technique (19 versus 43, <i>p</i> = 0.024). Thus, the application of the coaxial technique could improve diagnostic accuracy in patients with small and deep lung lesions, and could reduce the risk of pneumothorax. The combined use of cutting needles with coaxial needles is the preferred technique for performing percutaneous CT-guided lung biopsies.</p></div

The diagnostic yields in different subgroups.

<p>The diagnostic yields in different subgroups.</p

Comparison of patients, lesions, and procedural variables in both groups.

<p>Comparison of patients, lesions, and procedural variables in both groups.</p

The diagnostic yields of the two groups.

<p>The diagnostic yields of the two groups.</p

Rat LIPC sera reduced MDA and increased the activities of antioxidases in H₂O₂-injured HUVECs.

<p>Medium MDA concentration was measured spectrophotometrically at 532 nm. The activities of CAT, GSH-Px and total SOD were analyzed spectrophotometrically at 240 nm, 340 nm and 550 nm respectively. All data are expressed as mean ± SEM, n = 6, *<i>P</i> < 0.05, **<i>P</i> < 0.01 vs model.</p