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In response to comments, the USPSTF also added information about the currently low uptake of lung cancer screening and data on the effect of the current recommendation on eligibility for screening in Latinx/Hispanic persons. Last, the USPSTF added and updated resources and website links in the Additional Tools and Resources section.
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Six-week-old female BALB/c nude mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences and Technology (Shanghai, China). All animals were housed and maintained in specific pathogen-free conditions according to the recommendation of Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with strict accordance with protocols approved by the Institutional Animal Care and Use Committee of Guizhou Medical University.
Next, we analyzed the expression level of HDACs in control, USP38 knockdown, and overexpressing HCT116 cells to determine which HDAC is responsible for USP38 mediated histone modification. However, our results showed that the protein levels of five HDACs (HDAC1, HDAC2, HDAC3, HDAC4, and HDAC8) were not changed in USP38 knockdown and overexpressing cells (Fig. 5a). We then immunoprecipitated (IP) USP38 with HDACs to detect whether any HDAC is associated with USP38. Interestingly, our results showed that HDAC3, but not other HDACs, was specifically associated with USP38 (Fig. 5b, c). Since, USP38 is a potential deubiquitinase, we examined the ubiquitination level of HDAC3 in control and USP38 overexpressing HCT116 cells. Importantly, we found that USP38 overexpression significantly decreased the ubiquitination level of HDAC3 but not HDAC1 and had no effect on the overall protein level of HDAC3 and HDAC1 (Fig. 5d). Moreover, the lysine 63 ubiquitin chain but not the lysine 48 ubiquitin chain of HDAC3 were specifically cleaved by USP38 (Fig. 5e) which explains the unchanged overall HDAC3 protein level. To further prove that USP38 functions as a DUB in removing the K63-linked ubiquitination of HDAC3, we generated a DUB mutant of USP38 (C454S/H857A/D918N), which lacks the DUB activity16. We found that the ubiquitination of HDAC3 was erased by WT USP38 but not the enzyme activity mutant of USP38 (Fig. 5f). In order to identify the K63 ubiquitination site on HDAC3, we generated four K to R mutants of HDAC3 (K44R, K83R, K121R, and K233R). WT HDAC3 and four KR mutant HDAC3 were transfected with HA-Ub K63 only (all lysine sites on Ubiquitin were mutated to arginine, except for lysine at 63, the HA-Ub K63 only plasmid could only generate Lys-63 ubiquitination chain) plasmids. We found that K121R mutant of HDAC3 was unable to form K63 ubiquitination chain, suggesting that lysine 121 may be the K63 ubiquitination site on HDAC3 (Fig. 5g). Then, we detected HDAC3 activity in HDAC3 depleted cells after reconstructed HDAC3 expression with WT or K121R mutant HDAC3. Our results showed that HDAC3 K121R mutant cells exhibited lower HDAC3 activity compared with WT HDAC3 (Fig. 5h). K121R mutant also showed higher level of H3K27ac compared with WT HDAC3 (Fig. 5i). These results indicated that K63 ubiquitination of HDAC3 was required for the deacetylation activity of HDAC3. Next, we performed a ChIP assay to illustrate the binding infinity of HDAC3 and the promoter of CD133. As shown in Fig. 5j, the binding of HDAC3 to the promoter of CD133 was affected by neither USP38 knockdown nor USP38 overexpression. Taken together, K121 of HDAC3 is required for its K63-linked ubiquitination of HDAC3 and the deacetylation activity of HDAC3. Hence, our data indicated that USP38 functions as a deubiquitinase of HDAC3 to modulate the acetylation of histones and regulate gene expression.
All the human colorectal cancer specimens were collected in Affiliated Hospital of Guizhou Medical University. The use of patient samples was approved by the Ethics Committee of Guizhou Medical University (Approval no. 2018-123-01) and informed consent was obtained from the patients. All animals were housed and maintained in specific pathogen-free conditions according to the recommendation of Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with strict accordance with protocols approved by the Ethics Committee of Guizhou Medical University (Approval no.1900628)
The ciliopathies are a group of phenotypically overlapping disorders caused by structural or functional defects in the primary cilium. Although disruption of numerous signaling pathways and cellular trafficking events have been implicated in ciliary pathology, treatment options for affected individuals remain limited. Here, we performed a genome-wide RNAi (RNA interference) screen to identify genetic suppressors of BBS4, one of the genes mutated in Bardet-Biedl syndrome (BBS). We discovered 10 genes that, when silenced, ameliorate BBS4-dependent pathology. One of these encodes USP35, a negative regulator of the ubiquitin proteasome system, suggesting that inhibition of a deubiquitinase, and subsequent facilitation of the clearance of signaling components, might ameliorate BBS-relevant phenotypes. Testing of this hypothesis in transient and stable zebrafish genetic models showed this posit to be true; suppression or ablation of usp35 ameliorated hallmark ciliopathy defects including impaired convergent extension (CE), renal tubule convolution, and retinal degeneration with concomitant clearance of effectors such as β-catenin and rhodopsin. Together, our findings reinforce a direct link between proteasome-dependent degradation and ciliopathies and suggest that augmentation of this system might offer a rational path to novel therapeutic modalities.
An prominent role of primary cilium is to act as a signaling center (18, 19), facilitating the communication between extra- and intracellular signaling effector molecules during development and disease prognosis (2). To date, this organelle has been linked to several paracrine pathways crucial for development, including Wnt, Shh, and Notch (20). We posited that the identification of genes whose suppression could rescue aberrant signal transduction caused by ciliary dysfunction might have therapeutic value. To identify such targets, we designed a genome-wide RNAi screening paradigm, with the aim of isolating genes whose suppression can rescue hyperactive Wnt/β-catenin (β-cat) signaling in the absence of BBS4 (a ciliary gene mutated in patients with BBS, an archetypal ciliopathy; refs. 21, 22).
AXIN2 expression is a direct target of Wnt/β-cat signaling (26) and has been used to evaluate the activation of Wnt/β-cat signaling (27). Consistent with the Wnt-reporter assay, BBS4 depletion enhances AXIN2 expression significantly (Supplemental Figure 2). Therefore, to validate the 29 hits, we transfected siRNA of each of the 29 genes into the same cell type used for the primary screen and performed quantitative PCR (qPCR) to quantify AXIN2 message. In the context of BBS4 knockdown, suppression of 14 of 29 genes led to significant reduction of AXIN2 message in comparison with control siRNA (Figure 1B). Although the roles of some of these genes are unclear, the identified hits are involved in different cellular mechanisms (Supplemental Table 1). These results suggest the link of ciliopathies to both existing and potentially new cellular mechanisms. Given that these hits are identified through in vitro Wnt/β-cat reporter, we next asked whether the rescue of hyperactive Wnt/β-cat signaling can also be observed in vivo. During early development, hyperactivation of the Wnt/β-cat pathway perturbs planar cell polarity, impairing proper CE (23, 28). Therefore, we investigated the in vivo effects of the 14 candidate genes by assessing their effects on the CE phenotype observed in bbs4 zebrafish morphants (20, 23). We designed morpholinos (MO) targeting 11 of the 14 candidates (DTX1, PITPNM2, ENTPD6, C14orf166B, TEX36, ZIC1, PTMA, ENGASE, ENPP7, TDRD12, and USP35) and guide RNA targeting 2 candidates (DRD5 and PCF11; lack of appropriate splicing blocker site) to perturb the expression of candidate genes (RHOXF1 was excluded from further analysis due to the lack of a zebrafish ortholog) (Supplemental Figure 3). We scored the phenotypic effects resulting from suppression of these 13 candidates by CE analysis; we found that suppression of 10 of 13 rescued significantly the CE defects of bbs4 morphants (Figure 1C, Figure 2A, and Supplemental Figure 4).
The Python interpreter and the extensive standard library are freely availablein source or binary form for all major platforms from the Python web site, , and may be freely distributed. The same site alsocontains distributions of and pointers to many free third party Python modules,programs and tools, and additional documentation. 2b1af7f3a8