Growing evidence has highlighted the immune response as an important feature of carcinogenesis and therapeutic efficacy in clear cell renal cell carcinoma (ccRCC). This study categorized ccRCC cases into high and low score groups based on their immune/stromal scores generated by the ESTIMATE algorithm, and identified an association between these scores and prognosis. Differentially expressed tumor environment (TME)-related genes extracted from common upregulated components in immune and stromal scores were described using functional annotations and protein-protein interaction (PPI) networks. Most PPIs were selected for further prognostic investigation. Many additional previously neglected signatures, including AGPAT9, AQP7, HMGCS2, KLF15, MLXIPL, PPARGC1A, exhibited significant prognostic potential. In addition, multivariate Cox analysis indicated that MIXIPL and PPARGC1A were the most significant prognostic signatures, and were closely related to immune infiltration in TCGA cohort. External prognostic validation of MIXIPL and PPARGC1A was undertaken in 380 ccRCC cases from a real-world cohort. These findings indicate the relevance of monitoring and manipulation of the microenvironment for ccRCC prognosis and precision immunotherapy.

The ERCC1-XPF heterodimer is a structure-specific endonuclease and plays multiple roles in various DNA repair pathways including nucleotide excision repair and also telomere maintenance. The dimer formation, which is mediated by their C-terminal helix-hairpin-helix regions, is essential for their endonuclease activity as well as the stability of each protein. However, the detailed mechanism of how a cellular level of ERCC1-XPF is regulated still remains elusive. Here, we report the identification of DDB1- and CUL4-associated factor 7 (DCAF7, also known as WDR68/HAN11) as a novel interacting protein of ERCC1-XPF by mass spectrometry after tandem purification. Immunoprecipitation experiments confirmed their interaction and suggested dominant association of DCAF7 with XPF but not ERCC1. Interestingly, siRNA-mediated knockdown of DCAF7, but not DDB1, attenuated the cellular level of ERCC1-XPF, which is partly dependent on proteasome. The depletion of TCP1α, one of components of the molecular chaperon TRiC/CCT known to interact with DCAF7 and promote its folding, also reduced ERCC1-XPF level. Finally, we show that the depletion of DCAF7 causes inefficient repair of UV-induced (6-4) photoproducts, which can be rescued by ectopic overexpression of XPF or ERCC1-XPF. Altogether, our results strongly suggest that DCAF7 is a novel regulator of ERCC1-XPF protein level and cellular nucleotide excision repair activity.

High grade serous carcinoma (HGSC) is the most common and most aggressive, subtype of epithelial ovarian cancer. It presents as advanced stage disease with poor prognosis. Recent pathological evidence strongly suggests HGSC arises from the fallopian tube via the precursor lesion; serous tubal intraepithelial carcinoma (STIC). However, further definition of the molecular evolution of HGSC has major implications for both clinical management and research. This study aims to more clearly define the molecular pathogenesis of HGSC.

Viral and endogenous double-stranded RNA (dsRNA) is a potent trigger for programmed RNA degradation by the 2-5A/RNase L complex in cells of all mammals. This 2-5A-mediated decay (2-5AMD) is a conserved stress response switching global protein synthesis from homeostasis to production of interferons (IFNs). To understand this mechanism, we examined 2-5AMD in human cells and found that it triggers polysome collapse characteristic of inhibited translation initiation. We determined that translation initiation complexes and ribosomes purified from translation-arrested cells remain functional. However, spike-in RNA sequencing (RNA-seq) revealed cell-wide decay of basal mRNAs accompanied by rapid accumulation of mRNAs encoding innate immune proteins. Our data attribute this 2-5AMD evasion to better stability of defense mRNAs and positive feedback in the IFN response amplified by RNase L-resistant molecules. We conclude that 2-5AMD and transcription act in concert to refill mammalian cells with defense mRNAs, thereby "prioritizing" the synthesis of innate immune proteins.

Flexible heteroarotinoids (Flex-Hets) are compounds with promising anti-cancer activities. SHetA2, a first-generation Flex-Het, has been shown to inhibit the growth of cervical, head and neck, kidney, lung, ovarian, prostate, and breast cancers. However, SHetA2's high lipophilicity, limited selectivity, low oral bioavailability, and complicated synthesis has led to the development of second-generation compounds, such as 1-(1-(naphthalen-1-yl)ethyl)-3-(4-nitrophenyl) thiourea or SL-1-09. Results from our lab show that SL-1-09 exhibits anti-cancer activities against ERα+ and ERα- breast cancer cells at micromolar concentrations. SL-1-09 is a mixture of two enantiomers, R and S. The objective of this study was to further analyze these enantiomers to determine their individual anti-cancer activities. Cell cycle analysis demonstrated that the percentage of cells in S-phase is reduced significantly when breast cancer cell lines MCF-7, T47D and MDA-MB-453 cells are treated with 5.0 μM of the S enantiomer. Consistent with this finding, treatment of these cells with the S enantiomer resulted in lower expression levels of cell cycle proteins. Overall, our data indicate that the S enantiomer shows greater growth inhibitory effects than the R form against ERα+ (MCF7 and T47D) and ERα- (MDA-MB-453) breast cancer cells, suggesting that the activity observed in SL-1-09 is most likely due to the ability of the S enantiomer to block cell cycle progression.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

Tumor cell nucleus is the ultimate target of many first-line chemotherapeutics and therapeutic genes. However, nuclear drug delivery is always hampered by multiple intracellular obstacles especially low efficiency of cellular uptake and insufficient nuclear trafficking. It is urgent to establish novel nuclear drug delivery systems to simultaneously overcome barriers including cell membranes and nuclear envelope. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed to achieve enhanced intracellular and intranuclear drug delivery. A biomimetic peptide (SVS-1), derived from antimicrobial peptides, which was reported to efficiently penetrate cell membranes and translocate rapidly into nucleus without decreasing cell viability, was conjugated to the HPMA copolymer backbone. The in vitro studies showed that SVS-1 could enhance the uptake and nuclei accumulation of HPMA copolymer by 4.1 and 7.0-fold on human cervical cancer cells (HeLa) separately compared with corresponding non-SVS-1 modified HPMA copolymers (P-DOX). This also transferred to greater DNA damage, more apoptosis and superior cytotoxicity (2.4-fold) of doxorubicin which was chosen as the model drug and attached to SVS-1 modified HPMA copolymer (SVS-1-P-DOX). Furthermore, the in vivo investigation revealed that compared with free doxorubicin, SVS-1-P-DOX not only showed prolonged blood circulation and preferential tumor accumulation, but also suppressed tumor growth more efficiently with tumor growth inhibition of 78.7% in HeLa tumor-bearing BALB/c nude mice without causing noticeable physiological change in major organs. These results demonstrated that the SVS-1 modification was a promising strategy for contemporaneously overcome cell membranes and nuclear envelope, which might provide new opportunities for constructing nucleus-targeted anticancer therapy.

Bergenin, a plant polyphenol, has been reported to lower the blood glucose level and ameliorate kidney function in streptozotocin (STZ)-induced diabetic rats. Herein, its protective effect on diabetic nephropathy (DN) was explored in view of extracellular matrix (ECM) generation in glomerular mesangial cells. Glomerular mesangial cells were treated with high glucose, and Q-PCR as well as western blot were used to determine the expression of ECM. To establish the participation and role of mammalian target of rapamycin (mTOR) and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in ECM generation, a combination of l-leucine (activator of mTOR) and Nrf2 shRNA transfection were performed, respectively. Moreover, a DN model was established in mice using high-glucose/high-fat diet and STZ. Bergenin impeded the generation of TGF-β1 and ECM, decreased the levels of intracellular superoxide anion and hydrogen peroxide, and increased the activity of antioxidant enzymes in the glomerular mesangial cells (HBZY-1 and HRMC cells) treated with high glucose. The inhibition of ECM generation by bergenin was dependent on the down-regulation of oxidative stress as confirmed via a superoxide overexpression system. The activation of Nrf2 was required for bergenin to inhibit the oxidative stress and ECM generation. Moreover, bergenin was found to inhibit the phosphorylation of mTOR, which is located at the upstream of Nrf2. Bergenin did not interfere with the expression of Nrf2 mRNA and Keap1 (the classic degradation control factor of Nrf2), but markedly inhibited the protein expression of the β-TrcP, an effect which could be abolished by l-leucine. In DN model mice, l-leucine diminished the ability of bergenin to reduce the levels of superoxide anion, hydrogen peroxide and ECM, which contributed to the eradication of the ameliorative effect of bergenin on nephropathy. Bergenin can inhibit glucose-induced ECM production in glomerular mesangial cells through the down-regulation of oxidative stress via the mTOR/β-TrcP/Nrf2 pathway, and it might be a candidate drug for the prevention and treatment of DN.

Several protein-RNA cross linking protocols have been established in recent years to delineate the molecular interaction of an RNA Binding Protein (RBP) and its target RNAs. However, functional dissection of the role of the RBP binding sites in modulating the post-transcriptional fate of the target RNA remains challenging. CRISPR/Cas9 genome editing system is being commonly employed to perturb both coding and noncoding regions in the genome. With the advancements in genome-scale CRISPR/Cas9 screens, it is now possible to not only perturb specific binding sites but also probe the global impact of protein-RNA interaction sites across cell types. Here, we present SliceIt (http://sliceit.soic.iupui.edu/), a database of in silico sgRNA (single guide RNA) library to facilitate conducting such high throughput screens. SliceIt comprises of ~4.8 million unique sgRNAs with an estimated range of 2-8 sgRNAs designed per RBP binding site, for eCLIP experiments of >100 RBPs in HepG2 and K562 cell lines from the ENCODE project. SliceIt provides a user friendly environment, developed using advanced search engine framework, Elasticsearch. It is available in both table and genome browser views facilitating the easy navigation of RBP binding sites, designed sgRNAs, exon expression levels across 53 human tissues along with prevalence of SNPs and GWAS hits on binding sites. Exon expression profiles enable examination of locus specific changes proximal to the binding sites. Users can also upload custom tracks of various file formats directly onto genome browser, to navigate additional genomic features in the genome and compare with other types of omics profiles. All the binding site-centric information is dynamically accessible via "search by gene", "search by coordinates" and "search by RBP" options and readily available to download. Validation of the sgRNA library in SliceIt was performed by selecting RBP binding sites in Lipt1 gene and designing sgRNAs. Effect of CRISPR/Cas9 perturbations on the selected binding sites in HepG2 cell line, was confirmed based on altered proximal exon expression levels using qPCR, further supporting the utility of the resource to design experiments for perturbing protein-RNA interaction networks. Thus, SliceIt provides a one-stop repertoire of guide RNA library to perturb RBP binding sites, along with several layers of functional information to design both low and high throughput CRISPR/Cas9 screens, for studying the phenotypes and diseases associated with RBP binding sites.

Endometritis, a common inflammation of the uterus, often causes severe damage to human and animal reproductive health. Polydatin is a polyphenol extracted from the rhizome of Polygonum cuspidatum that has anti-inflammatory and anti-oxidative effects. The purpose of this study was to investigate the underlying protective effects and mechanisms of polydatin against lipopolysaccharide (LPS)-induced endometritis in mice. The mouse model of endometritis was established by injection of LPS through the vagina. The uterine tissues of each group were gathered to analyze histopathological changes, inflammatory cytokine production, and the degree of activation of the NF-κB and Nrf2 signaling pathways. The myeloperoxidase (MPO) activity assay indicated that polydatin treatment significantly alleviated inflammatory cell infiltration in LPS-induced endometritis mice. Furthermore, polydatin treatment remarkably impeded the expression of TNF-α, IL-1β, and IL-6 by ELISA assay. Hematoxylin-eosin staining (H&E) showed that polydatin significantly decreased impairment of the uterus. In addition, polydatin was also found to suppress LPS-induced NF-κB activation in a dose-dependent manner. The expression of Nrf2 and HO-1 was enhanced by polydatin treatment. All the results suggest that polydatin helpfully alleviates LPS-induced endometritis by suppressing the NF-ĸB signaling pathway and activating the Nrf2 signaling pathway.

To describe and compare cytokines levels in clinically healthy sites of dental implants and natural teeth.

Variants in RCBTB1 have been implicated in inherited retinal disease (IRD). Here, we generated induced pluripotent stem cells (iPSCs) from a 45-year-old female IRD patient harbouring compound heterozygous mutations in the RCBTB1 gene. Episomal plasmids containing OCT4, SOX2, KLF4, MYCL, LIN28, shRNA for TP53 and mir302/367 microRNA were employed to conduct the reprogramming of primary dermal fibroblasts. These iPSC lines provide a useful model for further investigations on the pathophysiological role of mutations in the RCBTB1 gene in IRD.

Farnesoid X receptor (FXR) is a key regulator in charge of bile acid synthesis, transport, and metabolism. Activation of FXR represses bile acid synthesis and increases its excretion and transport, consequently protecting the liver functions. Thus, FXR is considered as a critical therapeutic target of cholestasis and nonalcoholic steatohepatitis. Herein, we isolated and identified fourteen new protostane-type triterpenoids (1-14) and four known analogues (15-18) from Alisma orientale, and finally constructed a small library of protostane-type triterpenoids (1-70) to investigate their structure-activity relationship with FXR, further leading to obtain compound 15 with potent agonistic activity against FXR (EC50 = 90 nM). Extensive in vitro investigation confirmed high efficacy of compound 15 against FXR in living cell, and revealed its underlying mechanism for FXR activation (amino acid residues Arg331 and Ser332) by molecular docking and site-directed mutagenesis technology.

As our research focus on anticancer drugs, two series of novel derivatives of Flexicaulin A (FA), an ent-kaurene diterpene, condensation with amino acid trifluoroacetate were synthesized, and their anti-proliferative activity against four human cancer cell lines (TE-1, MCF-7, A549 and MGC-803) were evaluated. Compared with FA, the anticancer activity and solubility of most derivatives were significantly improved. Among them, compound 6d had the best activity, and its IC50 value against Esophageal cancer cells (TE-1) was up to 0.75 μM. Subsequent cellular mechanism studies showed that compound 6d could inhibit the proliferation of cancer cells, the formation of cell colonies, and increase the level of ROS on TE-1 cells. In addition, 6d could up-regulate the expressions of SAPK/JNK pathway-associated proteins (p-ASK1, p-MKK4 and p-JNK) and pro-apoptotic proteins (Bak, Bad and Noxa), remarkably increase the ratio of Bax to Bcl-2 and activate Cleaved Caspase-3/9/PARP. These results indicate that compound 6d induces apoptosis through the ROS/JNK/Bcl-2 pathway and holds promising potential as an anti-proliferative agent.

For many people with advanced osteoarthritis, total knee replacement (TKR) is an effective treatment for relieving pain and improving function. Features of perioperative care may be associated with the adverse event of chronic pain 6 months or longer after surgery; effects may be direct, for example, through nerve damage or surgical complications, or indirect through adverse events. This systematic review aims to evaluate whether non-surgical perioperative interventions prevent long-term pain after TKR.

BACKGROUND This study assessed whether serum Golgi phosphoprotein 3 (GOLPH3) could be used as a biomarker for detecting bladder cancer. MATERIAL AND METHODS Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) assay were performed to measure GOLPH3 expression in serum and tissue samples, respectively, of bladder cancer patients. The associations of serum GOLPH3 expression with clinicopathological factors and the diagnostic accuracy were statistically evaluated using the chi-square test and receiver operating characteristic (ROC) curve analysis. RESULTS Compared with the healthy control group, serum GOLPH3 level was distinctly enhanced in bladder cancer patients (P<0.001). Moreover, compared to the non-malignant tissues, GOLPH3 showed positive expression in bladder cancer tissues. The abnormal GOLPH3 levels were tightly related to grade (P=0.018), tumor stage (P=0.000), lymph node status (P=0.030), and muscle invasion (P=0.012). ROC analysis showed that serum GOLPH3 exhibited a high diagnostic value to distinguish bladder cancer patients from healthy persons. The area under the ROC curve (AUC) was 0.948. The specificity and sensitivity were 92.5% and 83.8%, respectively. CONCLUSIONS GOLPH3 was highly expressed in bladder cancer patients and could be used as a diagnostic tool.

Meiosis is a conserved tenet of sexual reproduction in eukaryotes, yet this program is seemingly absent from many extant species. In the human fungal pathogen Candida albicans, mating of diploid cells generates tetraploid products that return to the diploid state via a non-meiotic process of depolyploidization known as concerted chromosome loss (CCL). Here, we report that recombination rates are more than three orders of magnitude higher during CCL than during normal mitotic growth. Furthermore, two conserved 'meiosis-specific' factors play central roles in CCL as SPO11 mediates DNA double-strand break formation while both SPO11 and REC8 regulate chromosome stability and promote inter-homolog recombination. Unexpectedly, SPO11 also promotes DNA repair and recombination during normal mitotic divisions. These results indicate that C. albicans CCL represents a 'parameiosis' that blurs the conventional boundaries between mitosis and meiosis. They also reveal parallels with depolyploidization in mammalian cells and provide potential insights into the evolution of meiosis.

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

Neuroimaging studies of the Psychomotor Vigilance Task (PVT) have revealed brain regions involved in attention lapses in sleep-deprived and well-rested adults. Those studies have focused on individual brain regions, rather than integrated brain networks, and have overlooked adolescence, a period of ongoing brain development and endemic short sleep. This study used functional MRI (fMRI) and a contemporary analytic approach to assess time-resolved peri-stimulus response of key brain networks when adolescents complete the PVT, and test for differences across attentive versus inattentive periods and after short sleep versus well-rested states. Healthy 14-17-year-olds underwent a within-subjects randomized protocol including 5-night spans of extended versus short sleep. PVT was performed during fMRI the morning after each sleep condition. Event-related independent component analysis (eICA) identified coactivating functional networks and corresponding time courses. Analysis of salient time course characteristics tested the effects of sleep condition, lapses, and their interaction. Seven eICA networks were identified supporting attention, executive control, motor, visual, and default-mode functions. Attention lapses, after either sleep manipulation, were accompanied by broadly increased response magnitudes post-stimulus and delayed peak responses in some networks. Well-circumscribed networks respond during the PVT in adolescents, with timing and intensity impacted by attentional lapses regardless of experimentally shortened or extended sleep.

In vitro assays and simulation technologies are powerful methodologies that can inform scientists of nanomaterial (NM) distribution and fate in humans or pre-clinical species. For small molecules, less animal data is often needed because there are a multitude of in vitro screening tools and simulation-based approaches to quantify uptake and deliver data that makes extrapolation to in vivo studies feasible. Small molecule simulations work because these materials often diffuse quickly and partition after reaching equilibrium shortly after dosing, but this cannot be applied to NMs. NMs interact with cells through energy dependent pathways, often taking hours or days to become fully internalized within the cellular environment. In vitro screening tools must capture these phenomena so that cell simulations built on mechanism-based models can deliver relationships between exposure dose and mechanistic biology, that is biology representative of fundamental processes involved in NM transport by cells (e.g. membrane adsorption and subsequent internalization). Here, we developed, validated, and applied the FORECAST method, a combination of a calibrated fluorescence assay (CF) with an artificial intelligence-based cell simulation to quantify rates descriptive of the time-dependent mechanistic biological interactions between NMs and individual cells. This work is expected to provide a means of extrapolation to pre-clinical or human biodistribution with cellular level resolution for NMs starting only from in vitro data.