An interlaboratory capillary zone electrophoresis-UV study of various monoclonal antibodies, instruments, and ε-aminocaproic acid lots.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The ε-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

Interdisciplinary treatment with implant-supported two-unit cantilever prosthesis for a patient with hypodontia: A clinical report.

A 21-year-old woman with multiple congenitally missing maxillary anterior teeth received interdisciplinary treatment to restore function and esthetics. The treatment was initiated with orthodontic treatment, followed by implant placement, bone and soft-tissue augmentation, and prosthetic treatment including a screw-retained implant-supported 2-unit cantilever fixed dental prosthesis.

The Functions of PCNA in Tumor Stemness and Invasion.

Invasion is the most prominent lethal feature of malignant cancer. However, how cell proliferation, another important feature of tumor development, is integrated with tumor invasion and the subsequent cell dissemination from primary tumors is not well understood. Proliferating cell nuclear antigen (PCNA) is essential for DNA replication in cancer cells. Loss of phosphorylation at tyrosine 211 (Y211) in PCNA (pY211-PCNA) mitigates PCNA function in proliferation, triggers replication fork arrest/collapse, which in turn sets off an anti-tumor inflammatory response, and suppresses distant metastasis. Here, we show that pY211-PCNA is important in stromal activation in tumor tissues. Loss of the phosphorylation resulted in reduced expression of mesenchymal proteins as well as tumor progenitor markers, and of the ability of invasion. Spontaneous mammary tumors that developed in mice lacking Y211 phosphorylation contained fewer tumor-initiating cells compared to tumors in wild-type mice. Our study demonstrates a novel function of PCNA as an essential factor for maintaining cancer stemness through Y211 phosphorylation.

Validation of a shape-optimized 2-unit cantilevered inlay-retained fiber-reinforced resin-bonded dental prosthesis.

STATEMENT OF PROBLEM: Current designs of fiber-reinforced composite (FRC) resin-bonded fixed dental prostheses (RBFDPs) have a limited lifespan, failing mainly through veneer-fiber delamination, debonding, and fracture. PURPOSE: The purpose of this in vitro study was to validate a new inlay-retained 2-unit cantilevered RBFDP with an optimized cavity and fiber layout proposed in a previous study by using simulated occlusal loading. MATERIAL AND METHODS: Two groups of specimens (n=20), 1 with and 1 without glass fibers, were used to test the influence of the cavity design and that of the fiber layout on their load capacity, respectively. The specimens without fibers were directly cut from a resin-ceramic block by using a computer-aided manufacturing system, while those with fibers were manually fabricated with unidirectional glass fibers and composite resin in a silicone mold. The specimens with and without fibers were attached to abutments made of the same resin-ceramic with a cyanoacrylate-based adhesive and a resin-based dental cement, respectively. An increasing compressive load was applied on the mesial fossa of the premolar pontic until failure. Cracking in the specimens during loading was monitored with a 2-channel acoustic emission (AE) system. RESULTS: All the specimens without fiber reinforcement debonded from the abutments. Those using the optimized shovel-shaped cavity design had a mean ±standard deviation failure load (50.0 ±17.3 N) that was 193% higher than that of those with the conventional step-box design (17.1 ±6.2 N; P<.001). No significant difference was found between the groups for the mean number of AE events per specimen (step-box: 49 ±34 versus shovel-shaped: 63 ±34; P=.427), the mean amplitude of each event (58.4 ±1.3 dB versus 59.5 ±2.4 dB; P=.299), or the mean time to failure (283.2 ±122.3 seconds versus 297.5 ±66.7 seconds; P=.798). Between the groups of specimens with reinforcing fibers, the mean failure load of the conventional design was approximately half that of the optimized one. Again, no significant difference was found for the mean number of AE events per specimen (conventional: 28 ±18 versus optimized: 52 ±53; P=.248) or the mean amplitude for each AE event (64.9 ±4.2 dB versus 61.7 ±5.2 dB; P=.187). The connectors of 8 fiber-reinforced specimens with the conventional design fractured; the other 2 debonded from the abutments. Half of the shape-optimized fiber-reinforced specimens had fractured abutments, but the cantilevers remained intact, 4 specimens fractured at the connector, and only 1 debonded from its abutment. CONCLUSIONS: The shape-optimized 2-unit cantilevered FRC RBFDP had a higher load capacity than the conventional design.

Ribonuclease 7-driven activation of ROS1 is a potential therapeutic target in hepatocellular carcinoma.

BACKGROUND & AIMS: There are currently limited therapeutic options for hepatocellular carcinoma (HCC), particularly when it is diagnosed at advanced stages. Herein, we examined the pathophysiological role of ROS1 and assessed the utility of ROS1-targeted therapy for the treatment of HCC. METHODS: Recombinant ribonucleases (RNases) were purified, and the ligand-receptor relationship between RNase7 and ROS1 was validated in HCC cell lines by Duolink, immunofluorescence, and immunoprecipitation assays. Potential interacting residues between ROS1 and RNase7 were predicted using a protein-protein docking approach. The oncogenic function of RNase7 was analyzed by cell proliferation, migration and invasion assays, and a xenograft mouse model. The efficacy of anti-ROS1 inhibitor treatment was evaluated in patient-derived xenograft (PDX) and orthotopic models. Two independent patient cohorts were analyzed to evaluate the pathological relevance of RNase7/ROS1. RESULTS: RNase7 associated with ROS1's N3-P2 domain and promoted ROS1-mediated oncogenic transformation. Patients with HCC exhibited elevated plasma RNase7 levels compared with healthy individuals. High ROS1 and RNase7 expression were strongly associated with poor prognosis in patients with HCC. In both HCC PDX and orthotopic mouse models, ROS1 inhibitor treatment markedly suppressed RNase7-induced tumorigenesis, leading to decreased plasma RNase7 levels and tumor shrinkage in mice. CONCLUSIONS: RNase7 serves as a high-affinity ligand for ROS1. Plasma RNase7 could be used as a biomarker to identify patients with HCC who may benefit from anti-ROS1 treatment. LAY SUMMARY: Receptor tyrosine kinases are known to be involved in tumorigenesis and have been targeted therapeutically for a number of cancers, including hepatocellular carcinoma. ROS1 is the only such receptor with kinase activity whose ligand has not been identified. Herein, we show that RNase7 acts as a ligand to activate ROS1 signaling. This has important pathophysiological and therapeutic implications. Anti-ROS1 inhibitors could be used to treatment patients with hepatocellular carcinoma and high RNase7 levels.

Imatinib (STI571) Inhibits the Expression of Angiotensin-Converting Enzyme 2 and Cell Entry of the SARS-CoV-2-Derived Pseudotyped Viral Particles.

A group of clinically approved cancer therapeutic tyrosine kinase inhibitors was screened to test their effects on the expression of angiotensin-converting enzyme 2 (ACE2), the cell surface receptor for SARS-CoV-2. Here, we show that the receptor tyrosine kinase inhibitor imatinib (also known as STI571, Gleevec) can inhibit the expression of the endogenous ACE2 gene at both the transcript and protein levels. Treatment with imatinib resulted in inhibition of cell entry of the viral pseudoparticles (Vpps) in cell culture. In FVB mice orally fed imatinib, tissue expression of ACE2 was reduced, specifically in the lungs and renal tubules, but not in the parenchyma of other organs such as the heart and intestine. Our finding suggests that receptor tyrosine kinases play a role in COVID-19 infection and can be therapeutic targets with combined treatments of the best conventional care of COVID-19.

Long noncoding RNA HOTAIR promotes invasion of breast cancer cells through chondroitin sulfotransferase CHST15.

The long noncoding RNA HOTAIR plays significant roles in promoting cancer metastasis. However, how it conveys an invasive advantage in cancer cells is not clear. Here we identify the chondroitin sulfotransferase CHST15 (GalNAc4S-6ST) as a novel HOX transcript antisense intergenic RNA (HOTAIR) target gene using RNA profiling and show that CHST15 is required for HOTAIR-mediated invasiveness in breast cancer cells. CHST15 catalyzes sulfation of the C6 hydroxyl group of the N-acetyl galactosamine 4-sulfate moiety in chondroitin sulfate to form the 4,6-disulfated chondroitin sulfate variant known as the CS-E isoform. We show that HOTAIR is necessary and sufficient for CHST15 transcript expression. Inhibition of CHST15 by RNA interference abolished cell invasion promoted by HOTAIR but not on HOTAIR-mediated migratory activity. Conversely, reconstitution of CHST15 expression rescued the invasive activity of HOTAIR-depleted cells. In corroboration with this mechanism, blocking cell surface chondroitin sulfate using a pan-CS antibody or an antibody specifically recognizes the CS-E isoform significantly suppressed HOTAIR-induced invasion. Inhibition of CHST15 compromised tumorigenesis and metastasis in orthotopic breast cancer xenograft models. Furthermore, the expression of HOTAIR closely correlated with the level of CHST15 protein in primary as well as metastatic tumor lesions. Our results demonstrate a novel mechanism underlying the function of HOTAIR in tumor progression through programming the context of cell surface glycosaminoglycans. Our results further establish that the invasive and migratory activities downstream of HOTAIR are distinctly regulated, whereby CHST15 preferentially controls the arm of invasiveness. Thus, the HOTAIR-CHST15 axis may provide a new avenue toward novel therapeutic strategies and prognosis biomarkers for advanced breast cancer.

The exosomal compartment protects epidermal growth factor receptor from small molecule inhibitors.

Epidermal growth factor receptor (EGFR) plays a significant role in promoting breast cancer progression. However, targeting EGFR as a single treatment only resulted in moderate efficacy to the disease. The underlying mechanism of low responsiveness to EGFR inhibition remains largely unclear. Tumor-secreted extracellular vesicles (EVs) play a crucial role in mediating intercellular communication between tumor and stromal cells in local microenvironment and distant metastatic niche. Extracellular vesicles mediate cell-to-cell transfer of lipids, nucleic acids, and proteins. Although numerous recent studies have demonstrated exchanges of extracellular vesicles between cancer cells and the recipient cells contribute to tumor proliferation, invasion, and metastasis, yet little is known how the exosomal compartment responds to targeted therapies and their role in promoting drug resistance. In the current study we used a triple-negative breast cancer model to show that EV-encapsulated EGFR is protected from targeted inhibitors of EGFR and can trigger signaling pathway in recipient cancer cells, promoting proliferation and migration ability in vitro. Taken together, our study suggested a novel mechanism of drug resistance entailing the EV compartment, such as exosomes, as a target shelter which when released can signal for tumor promotion in the recipient cancer cells.

Echocardiographic strain in hypertrophic cardiomyopathy and hypertensive left ventricular hypertrophy.

BACKGROUND: The myocardial structure differs between secondary left ventricular hypertrophy (LVH) and hypertrophic cardiomyopathy (HCM). We investigated left ventricular function of these two types of hypertrophy using multilayer strain analysis with two-dimensional echocardiography. METHODS: Transthoracic echocardiography (Vivid-E9) was performed in 240 patients with preserved left ventricular ejection fraction (LVEF ≥50%) and with either HCM (n = 80, 63 men, age 49.8 ± 14.1 years), hypertensive LVH (n = 80, 63 men, age 51.4 ± 13.3 years) or normal blood pressure and left ventricular structure (n = 80, 63 men, 50.8 ± 12.4 years). Quantitative multilayer longitudinal strain (LS), circumferential strain (CS), and radial strain (RS) were analyzed. The ratio of endo-/epi-myocardial strain was calculated. RESULTS: Longitudinal strain was significantly (P < 0.001) lower in HCM patients than normal controls (15.2 ± 4.2% vs 23.1 ± 2.7%), especially in hypertrophic segments (14.5 ± 4.4% vs 17.2 ± 3.2% in nonhypertrophic segments, P < 0.01). LS was lower in patients with hypertensive LVH, similarly in all left ventricular segments (20.7 ± 3.7%, P < 0.001 vs controls). CS was lower in the mid- and epicardium (P < 0.01), but not endocardium in HCM (P = 0.4), and preserved in all myocardial layers in hypertensive LVH. The endo-/epi-myocardial ratios of both LS and CS were higher in HCM than hypertensive LVH (P < 0.01). RS was higher (P < 0.01) in HCM than hypertensive LVH and controls. Endocardial CS and global RS were correlated with LVEF (r ≥ 0.32, P < 0.01). CONCLUSIONS: Hypertrophic cardiomyopathy patients had marked reductions in LS and CS, whereas patients with hypertensive LVH had less reduction in LS and preserved CS. The increased endo-/epi-myocardial ratios of LS and CS may be useful in differentiating HCM from hypertensive LVH.

Shielding against Unfolding by Embedding Enzymes in Metal-Organic Frameworks via a de Novo Approach.

We show that an enzyme maintains its biological function under a wider range of conditions after being embedded in metal-organic framework (MOF) microcrystals via a de novo approach. This enhanced stability arises from confinement of the enzyme molecules in the mesoporous cavities in the MOFs, which reduces the structural mobility of enzyme molecules. We embedded catalase (CAT) into zeolitic imidazolate frameworks (ZIF-90 and ZIF-8), and then exposed both embedded CAT and free CAT to a denature reagent (i.e., urea) and high temperatures (i.e., 80 °C). The embedded CAT maintains its biological function in the decomposition of hydrogen peroxide even when exposed to 6 M urea and 80 °C, with apparent rate constants kobs (s-1) of 1.30 × 10-3 and 1.05 × 10-3, respectively, while free CAT shows undetectable activity. A fluorescence spectroscopy study shows that the structural conformation of the embedded CAT changes less under these denaturing conditions than free CAT.

Maternal diethylhexyl phthalate exposure affects adiposity and insulin tolerance in offspring in a PCNA-dependent manner.

The ubiquitous plasticizer, diethylhexyl phthalate (DEHP), is a known endocrine disruptor. However, DEHP exposure effects are not well understood. Changes in industrial and agricultural practices have resulted in increased prevalence of DEHP exposure and has coincided with the heightened occurrence of metabolic syndrome and obesity. DEHP and its metabolites are detected in the umbilical cord blood of newborns; however, the prenatal and perinatal effects of DEHP exposure have not been intensively studied. Previously, we discovered that phosphorylation (p) of proliferating cell nuclear antigen (PCNA) at tyrosine 114 (Y114) is required for adipogenesis and diet-induced obesity in mice. Here, we show the unique ability of DEHP to induce p-Y114 in PCNA in vitro. We also show that while DEHP promotes adipogenesis of wild type (WT) murine embryonic fibroblasts, mutation of Y114 to phenylalanine (Y114F) in PCNA blocked adipocyte differentiation. Given the induction of p-Y114 in PCNA by DEHP and the relationship to obesity, WT and Y114F PCNA mice were exposed to DEHP during gestation or lactation, followed by high fat diet feeding. Paradoxically, in utero exposure of Y114F PCNA females to DEHP led to a significant increase in body mass and was associated with augmented expression of PPARγ, a critical regulator of obesity, compared to WT controls. In utero exposure of WT mice to DEHP led to insulin sensitivity while Y114F mutation ablated this phenotype, indicating that PCNA is an important regulator of early DEHP exposure and ensuing metabolic phenotypes.

Imparting functionality to biocatalysts via embedding enzymes into nanoporous materials by a de novo approach: size-selective sheltering of catalase in metal-organic framework microcrystals.

We develop a new concept to impart new functions to biocatalysts by combining enzymes and metal-organic frameworks (MOFs). The proof-of-concept design is demonstrated by embedding catalase molecules into uniformly sized ZIF-90 crystals via a de novo approach. We have carried out electron microscopy, X-ray diffraction, nitrogen sorption, electrophoresis, thermogravimetric analysis, and confocal microscopy to confirm that the ~10 nm catalase molecules are embedded in 2 µm single-crystalline ZIF-90 crystals with ~5 wt % loading. Because catalase is immobilized and sheltered by the ZIF-90 crystals, the composites show activity in hydrogen peroxide degradation even in the presence of protease proteinase K.

Advances and prospects of biomarkers for immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) activate anti-cancer immunity by blocking T cell checkpoint molecules such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Although ICIs induce some durable responses in various cancer patients, they also have disadvantages, including low response rates, the potential for severe side effects, and high treatment costs. Therefore, selection of patients who can benefit from ICI treatment is critical, and identification of biomarkers is essential to improve the efficiency of ICIs. In this review, we provide updated information on established predictive biomarkers (tumor programmed death-ligand 1 [PD-L1] expression, DNA mismatch repair deficiency, microsatellite instability high, and tumor mutational burden) and potential biomarkers currently under investigation such as tumor-infiltrated and peripheral lymphocytes, gut microbiome, and signaling pathways related to DNA damage and antigen presentation. In particular, this review aims to summarize the current knowledge of biomarkers, discuss issues, and further explore future biomarkers.

Tannic acids and proanthocyanidins in tea inhibit SARS-CoV-2 variants infection.

The COVID-19 pandemic has caused hundreds million cases and millions death as well as continues to infect human life in the world since late of 2019. The breakthrough infection caused from mutation of SARS-CoV-2 is rising even the vaccinated population has been increasing. Currently, the severe threat posed by SARS-CoV-2 has been alleviated worldwide, and the situation has transitioned to coexisting with the virus. The dietary food with antiviral activities may improve to prevent virus infection for living with COVID-19 pandemic. Teas containing enriched phenolic ingredients such as tannins have been reported to be antitumor agents as well as be good inhibitors for coronavirus. This study developed a highly sensitive and selective ultra-high performance liquid chromatography-high resolution mass spectrometric method for quantification of tannic acids, a hydrolysable tannin, and proanthocyanidins, a condense tannin, in teas with different levels of fermentation. The in vitro pseudoviral particles (Vpp) infection assay was used to evaluate the inhibition activities of various teas. The results of current research demonstrate that the tannins in teas are effective inhibitors against infection of SARS-CoV-2 and its variants.

Targeting ALK averts ribonuclease 1-induced immunosuppression and enhances antitumor immunity in hepatocellular carcinoma.

Tumor-secreted factors contribute to the development of a microenvironment that facilitates the escape of cancer cells from immunotherapy. In this study, we conduct a retrospective comparison of the proteins secreted by hepatocellular carcinoma (HCC) cells in responders and non-responders among a cohort of ten patients who received Nivolumab (anti-PD-1 antibody). Our findings indicate that non-responders have a high abundance of secreted RNase1, which is associated with a poor prognosis in various cancer types. Furthermore, mice implanted with HCC cells that overexpress RNase1 exhibit immunosuppressive tumor microenvironments and diminished response to anti-PD-1 therapy. RNase1 induces the polarization of macrophages towards a tumor growth-promoting phenotype through activation of the anaplastic lymphoma kinase (ALK) signaling pathway. Targeting the RNase1/ALK axis reprograms the macrophage polarization, with increased CD8+ T- and Th1- cell recruitment. Moreover, simultaneous targeting of the checkpoint protein PD-1 unleashes cytotoxic CD8+ T-cell responses. Treatment utilizing both an ALK inhibitor and an anti-PD-1 antibody exhibits enhanced tumor regression and facilitates long-term immunity. Our study elucidates the role of RNase1 in mediating tumor resistance to immunotherapy and reveals an RNase1-mediated immunosuppressive tumor microenvironment, highlighting the potential of targeting RNase1 as a promising strategy for cancer immunotherapy in HCC.

Epidermal growth factor receptor protects proliferating cell nuclear antigen from cullin 4A protein-mediated proteolysis.

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA synthesis upon growth stimulation. It has been shown that phosphorylation of PCNA at Tyr-211 by the EGF receptor (EGFR) protects PCNA from polyubiquitylation and degradation, whereas blocking phosphorylation induces ubiquitylation-mediated degradation of the chromatin-bound, but not the -unbound, PCNA, and suppresses cell proliferation. However, the ubiquitin E3 ligase linking growth signaling to the proteolysis of PCNA and the underlying regulatory mechanism remain to be identified. Here we show that, in the absence of Tyr-211 phosphorylation, PCNA is subject to polyubiquitylation at Lys-164 by the CUL4A E3 ligase, resulting in the degradation of PCNA. Mutation of Lys-164 to arginine prevents PCNA ubiquitylation and rescues the degradation of the K164R/Y211F PCNA double mutant. Activation of EGFR inhibits the interaction of PCNA with CUL4A, whereas inhibition of EGFR leads to increased CUL4A-PCNA interaction and CUL4A-dependent ubiquitin-mediated degradation of PCNA. Substitution of endogenous PCNA with the Y211F mutant PCNA conveys enhanced sensitization to EGFR inhibition. Our findings identify CUL4A as the ubiquitin ligase linking the down-regulation of cell surface receptor tyrosine kinase to the nuclear DNA replication machinery in cancer cells.

Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet.

Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA(F/F)) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA(F/F) MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA(F/F) mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.

Water-based synthesis of zeolitic imidazolate framework-90 (ZIF-90) with a controllable particle size.

The ZIF code: ZIF-90 materials were successfully synthesized in an optimized water-based system. The particle size, ranging from micro- to nanoscales, could be controlled by different amounts of polyvinylpyrrolidone (PVP), Zn/imidazole-2-carboxaldehyde ratio and alcohol.

Tyrosine phosphorylation controls PCNA function through protein stability.

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.