Poly(ADP-ribosyl)ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates.

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.

Structural Insights into the Dynamic Process of ß2-Adrenergic Receptor Signaling.

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Correlating histone acetylation with nucleosome core particle dynamics and function.

Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails. However, a systematic, atomic resolution experimental investigation of how this epigenetic mark, focusing on one histone at a time, influences the structural dynamics of the nucleosome beyond the tails, and how this translates into accessibility of protein factors such as ligases and nucleases, has yet to be performed. Herein, using NMR spectroscopy of nucleosome core particles (NCPs), we evaluate the effects of acetylation of each histone on tail and core dynamics. We show that for histones H2B, H3, and H4, the histone core particle dynamics are little changed, even though the tails have increased amplitude motions. In contrast, significant increases to H2A dynamics are observed upon acetylation of this histone, with the docking domain and L1 loop particularly affected, correlating with increased susceptibility of NCPs to nuclease digestion and more robust ligation of nicked DNA. Dynamic light scattering experiments establish that acetylation decreases inter-NCP interactions in a histone-dependent manner and facilitates the development of a thermodynamic model for NCP stacking. Our data show that different acetylation patterns result in nuanced changes to NCP dynamics, modulating interactions with other protein factors, and ultimately controlling biological output.

PAGE-B incorporating moderate HBV DNA levels predicts risk of HCC among patients entering into HBeAg-positive chronic hepatitis B.

BACKGROUND & AIMS: Recent studies reported that moderate HBV DNA levels are significantly associated with hepatocellular carcinoma (HCC) risk in hepatitis B e antigen (HBeAg)-positive, non-cirrhotic patients with chronic hepatitis B (CHB). We aimed to develop and validate a new risk score to predict HCC development using baseline moderate HBV DNA levels in patients entering into HBeAg-positive CHB from chronic infection. METHODS: This multicenter cohort study recruited 3,585 HBeAg-positive, non-cirrhotic patients who started antiviral treatment with entecavir or tenofovir disoproxil fumarate at phase change into CHB from chronic infection in 23 tertiary university-affiliated hospitals of South Korea (2012-2020). A new HCC risk score (PAGED-B) was developed (training cohort, n = 2,367) based on multivariable Cox models. Internal validation using bootstrap sampling and external validation (validation cohort, n = 1,218) were performed. RESULTS: Sixty (1.7%) patients developed HCC (median follow-up, 5.4 years). In the training cohort, age, gender, platelets, diabetes and moderate HBV DNA levels (5.00-7.99 log10 IU/ml) were independently associated with HCC development; the PAGED-B score (based on these five predictors) showed a time-dependent AUROC of 0.81 for the prediction of HCC development at 5 years. In the validation cohort, the AUROC of PAGED-B was 0.85, significantly higher than for other risk scores (PAGE-B, mPAGE-B, CAMD, and REAL-B). When stratified by the PAGED-B score, the HCC risk was significantly higher in high-risk patients than in low-risk patients (sub-distribution hazard ratio = 8.43 in the training and 11.59 in the validation cohorts, all p <0.001). CONCLUSIONS: The newly established PAGED-B score may enable risk stratification for HCC at the time of transition into HBeAg-positive CHB. IMPACT AND IMPLICATIONS: In this study, we developed and validated a new risk score to predict hepatocellular carcinoma (HCC) development in patients entering into hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) from chronic infection. The newly established PAGED-B score, which included baseline moderate HBV DNA levels (5-8 log10 IU/ml), improved on the predictive performance of prior risk scores. Based on a patient's age, gender, diabetic status, platelet count, and moderate DNA levels (5-8 log10 IU/ml) at the phase change into CHB from chronic infection, the PAGED-B score represents a reliable and easily available risk score to predict HCC development during the first 5 years of antiviral treatment in HBeAg-positive patients entering into CHB. With a scoring range from 0 to 12 points, the PAGED-B score significantly differentiated the 5-year HCC risk: low <7 points and high ≥7 points.

Metabolomics and miRNA profiling reveals feature of gallbladder cancer-derived biliary extracellular vesicles.

BACKGROUND: Although there are several studies in the development of various human cancers, the role of exosomes is poorly understood in the progression of gallbladder cancer. This study aims to characterize the metabolic changes occurring in exosomes obtained from patients with gallbladder cancer compared with those from other gallbladder disease groups. METHODS: Biliary exosomes were isolated from healthy donors (n = 3) and from patients with gallbladder cancer (n = 3), gallbladder polyps (n = 4), or cholecystitis (n = 3) using a validated exosome isolation kit. Afterward, we performed miRNA profiling and untargeted metabolomic analysis of the exosomes. The results were validated by integrating the results of the miRNA and metabolomic analyses. RESULTS: The gallbladder cancer group exhibited a significant reduction in the levels of multiple unsaturated phosphatidylethanolamines and phosphatidylcholines compared to the normal group, which resulted in the loss of exosome membrane integrity. Additionally, the gallbladder cancer group demonstrated significant overexpression of miR-181c and palmitic acid, and decreased levels of conjugated deoxycholic acid, all of which are strongly associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: Our findings demonstrate that the contents of exosomes are disease-specific, particularly in gallbladder cancer, and that altered metabolites convey critical information regarding their phenotype. We believe that our metabolomic and miRNA profiling results may provide important insights into the development of gallbladder cancer.

Interaction hot spots for phase separation revealed by NMR studies of a CAPRIN1 condensed phase.

The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. While computational and theoretical approaches have provided significant insights into IDR phase behavior, establishing the critical interactions that govern condensation with atomic resolution through experiment is more difficult, given the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states. NMR can be a valuable method, but the dynamic and viscous nature of condensed IDRs presents challenges. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in stress granules, P bodies, and messenger RNA transport granules, we have developed and applied a variety of NMR methods for studies of condensed IDR states to provide insights into interactions driving and modulating phase separation. We identify ATP interactions with CAPRIN1 that can enhance or reduce phase separation. We also quantify specific side-chain and backbone interactions within condensed CAPRIN1 that define critical sequences for phase separation and that are reduced by O-GlcNAcylation known to occur during cell cycle and stress. This expanded NMR toolkit that has been developed for characterizing IDR condensates has generated detailed interaction information relevant for understanding CAPRIN1 biology and informing general models of phase separation, with significant potential future applications to illuminate dynamic structure-function relationships in other biological condensates.

Pneumonia Prevalence Upon Chest Radiography According to Vaccination Status Among Patients Under 50 Years of Age With Coronavirus Disease 2019.

BACKGROUND: Coronavirus disease 2019 (COVID-19) vaccination is effective in preventing the disease transmission and progression. However, the relatively mild disease course of the omicron variant and the decrease in antibodies over time after vaccination raise questions about the effectiveness of vaccination, especially in young people. We compared the prevalence of pneumonia and chest X-ray severity score according to vaccination status among patients < 50 years old with COVID-19. METHODS: From January 17 to March 17, 2022, 579 patients with COVID-19, who were < 50 years old and had a known vaccination history in our institution, were all included in this study. All patients underwent initial chest radiography, and follow-up chest radiographs were obtained every two days until discharge. Pneumonia was scored from the radiographs using the Brixia scoring system. The scores of the six lung zones were added for a total score ranging from 0 to 18. Patients were divided into four groups according to 10-year age intervals. Differences between groups were analyzed using the χ² or Fisher's exact tests for categorical variables and the Kruskal-Wallis test or analysis of variance for continuous variables. RESULTS: Among patients aged 12-19 years, the prevalence of pneumonia did not differ depending on vaccination status (non-vaccinated vs. vaccinated, 1/47 [2.1%] vs. 1/18 [5.6%]; P = 0.577). Among patients in their 20s, the prevalence of pneumonia was significantly higher among non-vaccinated patients than among vaccinated patients (8/28, 28.6% vs. 7/138, 5.1%, P < 0.001), similar to patients in their 40s (32/52 [61.5%] vs. 18/138 [13.0%]; P < 0.001). The chest X-ray severity score was also significantly higher in non-vaccinated patients than that in vaccinated patients in their 20s to their 40s (P < 0.001), but not among patients aged 12-19 years (P = 0.678). CONCLUSION: In patients aged 20-49 years, vaccinated patients had a significantly lower prevalence of pneumonia and chest X-ray severity score than non-vaccinated patients.

Suprapatellar intramedullary nail combined with screw fixation has comparable surgical outcomes to minimally invasive locking plate fixation in ipsilateral tibial plateau and shaft fractures.

PURPOSE: To compare union rate, union time, alignment, and complication rate in ipsilateral tibia plateau and shaft fractures treated via suprapatellar intramedullary nailing with screw fixation and minimally invasive locking plate fixation. MATERIALS AND METHODS: A retrospective study was conducted on 48 patients who underwent minimally invasive plate fixation (n = 35) or suprapatellar intramedullary nailing with screw fixation (n = 13), for the treatment of ipsilateral tibial plateau and shaft fractures with at least 1-year follow-up. Union rate, union time, radiologic alignment, and complication rate such as malalignment, nonunion, and fracture-related infection (FRI) were investigated. RESULTS: Demographic data were not different between the two groups. Coronal plane alignment was 0.17 ± 4.23 in the plate group and -0.48 ± 4.17 in the intramedullary nail group (p = 0.637). Sagittal plane alignment was -0.13 ± 5.20 in the plate group and -1.50 ± 4.01 in the suprapatellar intramedullary nail group (p = 0.313). Coronal and sagittal malalignment recorded equal results: (p > 0.99), FRI (p = 0.602), nonunion and union times recorded (p = 0.656) and (p = 0.683, 0.829), respectively, and showed no significant difference between the two groups. CONCLUSION: Suprapatellar intramedullary nailing with screw fixation had similar surgical outcomes with minimally invasive locking plate fixation in ipsilateral tibial plateau and shaft fractures in terms of union rate, union time, alignment, and complication rate. Thus, frequent use of intramedullary nailing combined with screw fixation is anticipated in patients with ipsilateral tibial plateau and shaft fractures when the soft tissue condition is not desirable. LEVEL OF EVIDENCE: Level III.

Closed reduction and internal fixation for tillaux fractures, based on pre-operative three-dimensional computed tomography.

OBJECTIVE: This study aimed to propose treatment protocol and identify patterns of tillaux fractures using three-dimensional (3D) computed tomography (CT) analysis and to describe an effective reduction technique. METHODS: Forty-two juvenile patients with tillaux fractures were evaluated with 3D-CT scan for fracture displacement pattern and received surgical treatment. Tillaux fragment was reduced by pushing the superomedial quadrant part of the fragment slightly downward towards the ankle joint from anterolateral to posteromedial through 5-mm skin incisions with mosquito forceps. A 4.0 cannulated screw was subsequently inserted from the anterolateral to the posteromedial side parallel to the ankle joint. We analysed the distance and direction of fracture displacement with 3D-CT before the surgery. Pre-operative and post-operative plain radiographs were evaluated. RESULTS: Pre-operative 3D-CT analysis revealed a common fracture pattern, varus tilt, and external rotation of fragment. We achieved satisfactory reduction with residual fracture gaps less than 2 mm in 42 cases. Two cases had a 13-mm anterior gap that was reduced by mini-open reduction because of periosteal impingement. No significant clinical complications were found. CONCLUSION: The closed reduction technique developed based on the fracture pattern identified by 3D-CT anatomical analysis is safe and effective in treating tillaux fractures.

Association of Physical Activity With Risk of Liver Fibrosis, Sarcopenia, and Cardiovascular Disease in Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: International guidelines recommend physical activity for subjects with nonalcoholic fatty liver disease (NAFLD). This study investigated the association of physical activity with risk of liver fibrosis, sarcopenia, and cardiovascular disease (CVD) in NAFLD. METHODS: In this multicenter, retrospective study, 11,690 NAFLD subjects who underwent a health screening program and were assessed for physical activity (metabolic equivalent task [MET]-min/week) between 2014 and 2020 were recruited. Liver fibrosis was assessed by using the fibrosis-4 index, NAFLD fibrosis score, and FibroScan-AST score, sarcopenia by using multi-frequency bioelectric impedance analysis, and CVD risk by using atherosclerotic CVD (ASCVD) risk score, and coronary artery calcium (CAC) score were calculated. RESULTS: The prevalence of fibrosis, sarcopenia, high probability of ASCVD, and high CAC score significantly decreased with increasing quartiles of physical activity (all P for trend <.001). In a fully adjusted model, physical activity above 600 MET-min/week (≥third quartile) was independently associated with a reduced risk of fibrosis (adjusted odds ratio [aOR] = 0.59; 95% confidence interval [CI], 0.40-0.86), sarcopenia (aOR = 0.72; 95% CI, 0.58-0.88), high probability of ASCVD (aOR = 0.58; 95% CI, 0.46-0.73), and high CAC score (aOR = 0.32; 95% CI, 0.13-0.83; all P <.05). In addition, increasing amounts of physical activity were significantly associated with risk reduction between fibrosis, sarcopenia, and high probability of ASCVD (all P for trend <.001). In subjects with sarcopenic obesity or lean NAFLD, physical activity was also independently associated with reduced risk of fibrosis and high probability of ASCVD (all P <.05). CONCLUSIONS: Physical activity showed a protective effect against fibrosis, sarcopenia, and CVD in NAFLD.

Risk Stratification for Sarcopenic Obesity in Subjects With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: The impact of the severity of sarcopenic obesity (SO) in nonalcoholic fatty liver disease (NAFLD) on the risk of significant liver fibrosis or cardiovascular disease (CVD) remains unclear. We aimed to identify high-risk subjects with SO for significant liver fibrosis or CVD among subjects with SO and NAFLD. METHODS: This multicenter, retrospective study involved 23,889 subjects with NAFLD who underwent a health screening program (2014-2020). Sarcopenia was defined based on gender-specific sarcopenia index cutoff using multi-frequency bioelectric impedance analysis. High-risk subjects with SO were defined as those with significant liver fibrosis by fibrosis-4 index >2.67 or atherosclerotic CVD risk score >20%. Multivariable logistic regression analysis for identifying high-risk subjects with SO was performed in a cross-sectional cohort with SO, and further validation was performed in a longitudinal cohort. RESULTS: SO prevalence was 5.4% (n = 1297 of 23,889). Older age (unstandardized beta [ß] = 3.23; P < .001), male (ß = 1.66; P = .027), sarcopenia index (ß = -6.25; P = .019), and metabolic syndrome (ß = 1.75; P < .001) were significant risk factors for high-risk SO. Based on a high-risk SO screening model, high-risk subjects with SO had significantly higher odds of significant liver fibrosis (training: adjusted odds ratio [aOR], 3.72; validation: aOR, 2.38) or CVD (training: aOR, 5.20; validation: aOR, 3.71) than subjects without SO (all P < .001). In subgroup analyses, the cumulative incidence of significant liver fibrosis or CVD development was significantly higher in high-risk subjects with SO than in low-risk subjects with SO in a longitudinal cohort considering all-cause mortality and liver transplantation as competing risks (sub-distribution hazard ratio, 5.37; P < .001). CONCLUSION: The high-risk screening model may enable the identification of high-risk subjects with SO with NAFLD.

Optimum selection criteria for secondary cytoreductive surgery in patients with recurrent epithelial ovarian cancer: A multicenter study from the Gynecologic Oncology Research Investigators coLLaborAtion group (GORILLA-3001).

BACKGROUND: To identify those most likely to benefit from secondary cytoreductive surgery (SCS), we evaluated the survival outcomes and factors predictive of prognosis in patients with recurrent ovarian cancer. METHODS: We retrospectively reviewed the medical records of patients with recurrent ovarian cancer treated at five high-volume Korean hospitals between 2010 and 2021. Recurrence characteristics, treatment methods, and potential predictors of survival were compared between the chemotherapy and surgery groups. RESULTS: Among all 670 patients, 88.1% had initial stage III/IV disease, and 215 (32.1%) underwent SCS. Among patients who underwent SCS, only those who achieved complete resection exhibited improved survival. Even in patients with residual disease < 1 cm after SCS, we observed no significant survival benefit (p = 0.942). In the multivariate Cox analysis, residual disease at primary surgery, progression-free interval, recurrence sites (≤3 regions or limited carcinomatosis), ascites, and SCS were significant predictors of survival. Meanwhile, the only factor predictive of complete resection after SCS was recurrence sites (p < 0.001). CONCLUSIONS: The benefits of SCS appear to be exclusive to cases of complete resection. We propose limited regional platinum-sensitive recurrence (≤3 regions or limited carcinomatosis) without ascites as the optimum selection criteria for SCS.

Feasibility of Raman spectroscopic identification of gall bladder cancer using extracellular vesicles extracted from bile.

Extracellular vesicles (EVs), which are heterogeneous membrane-based vesicles with bilayer cell membrane structures, could be versatile biomarkers for the identification of diverse diseases including cancers. With this potential, this study has attempted the Raman spectroscopic identification of gall bladder (GB) cancer by directly measuring the EV solution extracted from human bile without further sample drying. For this purpose, bile samples were obtained from four normal individuals and 21 GB polyp, eight hepatocellular carcinoma (HCC), and five GB cancer patients, and EVs were extracted from each of the bile samples. The Raman peak shapes of the EVs extracted from the GB cancer samples, especially the relative intensities of peaks in the 1560-1340 cm-1 range, were dissimilar to those of the samples from the normal, GB polyp, and HCC groups. The intensity ratios of peaks at 1537 and 1453 cm-1 and at 1395 and 1359 cm-1 of the GB cancer samples were lower and higher, respectively, than those of the samples of the remaining three groups. The differences of peak intensity ratios were statistically significant based on the Mann-Whitney U test. DNA/RNA bases, amino acids, and bile salts contributed to the spectra of EVs, and their relative abundances seemed to vary according to the occurrence of GB cancer. The varied metabolite compositions and/or structures of EVs were successfully demonstrated by the dissimilar peak intensity ratios in the Raman spectra, thereby enabling the discrimination of GB cancer.

Electrocardiogram-based deep learning algorithm for the screening of obstructive coronary artery disease.

BACKGROUND: Information on electrocardiogram (ECG) has not been quantified in obstructive coronary artery disease (ObCAD), despite the deep learning (DL) algorithm being proposed as an effective diagnostic tool for acute myocardial infarction (AMI). Therefore, this study adopted a DL algorithm to suggest the screening of ObCAD from ECG. METHODS: ECG voltage-time traces within a week from coronary angiography (CAG) were extracted for the patients who received CAG for suspected CAD in a single tertiary hospital from 2008 to 2020. After separating the AMI group, those were classified into ObCAD and non-ObCAD groups based on the CAG results. A DL-based model adopting ResNet was built to extract information from ECG data in the patients with ObCAD relative to those with non-ObCAD, and compared the performance with AMI. Moreover, subgroup analysis was conducted using ECG patterns of computer-assisted ECG interpretation. RESULTS: The DL model demonstrated modest performance in suggesting the probability of ObCAD but excellent performance in detecting AMI. The AUC of the ObCAD model adopting 1D ResNet was 0.693 and 0.923 in detecting AMI. The accuracy, sensitivity, specificity, and F1 score of the DL model for screening ObCAD were 0.638, 0.639, 0.636, and 0.634, respectively, while the figures were up to 0.885, 0.769, 0.921, and 0.758 for detecting AMI, respectively. Subgroup analysis showed that the difference between normal and abnormal/borderline ECG groups was not notable. CONCLUSIONS: ECG-based DL model showed fair performance for assessing ObCAD and it may serve as an adjunct to the pre-test probability in patients with suspected ObCAD during the initial evaluation. With further refinement and evaluation, ECG coupled with the DL algorithm may provide potential front-line screening support in the resource-intensive diagnostic pathways.

Comparison of suprapatellar intramedullary nailing versus minimal invasive locked plating for proximal tibia fractures.

PURPOSE: To compare the radiological alignment, union time, union rate, and complication rate between suprapatellar intramedullary nails and minimally invasive locking plate fixation in the treatment of proximal tibial fractures. MATERIALS AND METHODS: We retrospectively analyzed 103 patients who underwent plate fixation (n = 50) or suprapatellar intramedullary nailing (n = 53) for proximal tibial fractures involving the meta-diaphyseal junction between November 2015 and October 2020 at our institution. The union rate, union time, radiologic alignments, and complications, such as malalignment, nonunion, and deep infection, were investigated. RESULTS: The demographic data did not differ between the plate and suprapatellar intramedullary nail groups. The alignment of the coronal plane was 0.24 ± 3.19 in the plate group and - 0.49 ± 2.22 in the intramedullary nail group (p = 0.196). Sagittal plane alignment was - 0.29 ± 4.97 in the plate group and 0.24 ± 4.12 in the intramedullary nail group (p = 0.571), and coronal malalignment (p = 0.196), sagittal malalignment (p = 0.57), deep infection (p = 0.264), nonunion (p = 0.695), union time (p = 0.329), and final union rate (p = 0.699) were not significantly different between groups. CONCLUSION: Compared with the minimally invasive locking compression plate group, the suprapatellar intramedullary nail group yielded comparable results in terms of radiological alignment and complications. Considering that proximal tibial fractures are associated with high-energy trauma and severe soft tissue damage, we believe that a suprapatellar intramedullary nail may be a good alternative. LEVEL OF EVIDENCE: Level III.

Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus.

In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.

Targeting transforming growth factor-ß2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer.

Transforming growth factor-beta (TGF-ß) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-ß2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-ß signaling by TGF-ß2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-ß2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-ß2. Moreover, TGF-ß2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-ß2 may be a promising target in the immunotherapeutic strategy of TNBC.

Overexpression of CYP11A1 recovers cell cycle distribution in renal cell carcinoma Caki-1.

BACKGROUND: Clear cell renal carcinoma is commonly known for its metastasis propensity to outspread to other organs and is asymptomatic in the early stage. Recent studies have shown that deficiencies in CYP11A1 expression can lead to fatal adrenal failure if left untreated and are associated with downstream regulation in various cancer types. However, the molecular mechanisms of CYP11A1 and kidney cancer proliferation remain unclear. METHODS: Normal and renal carcinoma cell lines (HEK293 and Caki-1) were transfected with plasmid encoding CYP11A1 to overexpress the P450scc protein. Cell cycle distribution was investigated using flow cytometry. The expression of proteins related to C-Raf/ERK/JNK/p38 signaling pathways was examined using western blot. RESULTS: We observed that CYP11A1 overexpression suppressed the cyclin B1 and cell-division cycle 2 expression while cyclin-dependent kinases 2 and 4 were unaffected. Cancer cell migration and invasion were suppressed along with epithelial-intermediate metastatic markers Snail and Vimentin. In addition, in CYP11A1-overexpressing Caki-1 cells, cdc2/cyclinB1 was downregulated while the phosphorylation of cdc25c, a G2/M arrest-related upstream signal, was increased. The intrinsic-mitochondrial apoptosis markers were not significantly altered. We also identified that the C-Raf/ERK/JNK/p38 pathway is an important pro-apoptotic mechanism in CYP11A1-overexpressing cell-based models. Our results suggest that CYP11A1 overexpression recovered the disturbed cell cycle arrest distribution in renal carcinoma cell line Caki-1 through G2/M arrest and C-Raf/ERK/JNK pathway. CONCLUSIONS: Our findings may suggest promising new therapeutic targets to suppress kidney cancer proliferation without affecting normal cells, eventually improving the survival of patients with cancer.

Clinical implications of neoadjuvant chemotherapy in advanced endometrial cancer: a multi-center retrospective cohort study.

BACKGROUND: The mainstay of endometrial cancer treatment is surgical resection of tumors and postoperative adjuvant treatment is recommended if necessary. However, there is no consensus on the management of unresectable metastatic endometrial cancer. This study aimed to assess the feasibility and effectiveness of neoadjuvant chemotherapy followed by interval debulking surgery (NAC-IDS) in unresectable, metastatic endometrial cancer. METHODS: From the endometrial cancer cohorts of four institutions in Korea, we identified patients with International Federation of Gynecology and Obstetrics stages IIIC-IVB endometrial cancer who received NAC-IDS between January 2008 and December 2020. Through a medical record review, we collected patients' clinicopathological data. Progression-free survival (PFS), overall survival (OS), and the factors affecting survival outcomes were analyzed. RESULTS: Overall, 32 patients were included with endometrioid (n = 18), serous (n = 5), carcinosarcoma (n = 6), and other histological types (n = 3). Among them, 28 (87.5%) patients had stage IVB disease. The most common neoadjuvant chemotherapy (NAC) regimen was paclitaxel-carboplatin (n = 25, 78.1%), which was administered for a median of six cycles. While 26 (81.3%) patients showed an objective response, two (6.3%) progressed despite NAC. At the time of interval debulking surgery (IDS), 23 (71.9%) patients achieved complete cytoreduction. During 31.0 months of the median follow-up, there were 23 recurrences and 11 deaths, corresponding to a median PFS of 19.7 months and a 3-year OS rate of 69.7%. In multivariate analyses, non-endometrioid histology and residual tumor after IDS were identified as independent poor prognostic factors for PFS (adjusted hazard ratio [HR], 7.322; P < 0.001 and 5.934; P = 0.001, respectively). Multivariate analysis for OS could not be conducted because of the small number of events, although non-endometrioid histology was the only factor associated with worse OS in univariate analysis (adjusted HR, 4.523; P = 0.032). CONCLUSIONS: NAC-IDS may be a treatment option for unresectable metastatic endometrial cancer. Tumor histology and the possibility of complete cytoreduction are the primary considerations for NAC-IDS.

A phase IA dose-escalation study of PHI-101, a new checkpoint kinase 2 inhibitor, for platinum-resistant recurrent ovarian cancer.

BACKGROUND: PHI-101 is an orally available, selective checkpoint kinase 2 (Chk2) inhibitor. PHI-101 has shown anti-tumour activity in ovarian cancer cell lines and impaired DNA repair pathways in preclinical experiments. Furthermore, the in vivo study suggests the synergistic effect of PHI-101 through combination with PARP inhibitors for ovarian cancer treatment. The primary objective of this study is to evaluate the safety and tolerability of PHI-101 in platinum-resistant recurrent ovarian cancer. METHODS: Chk2 inhibitor for Recurrent EpitheliAl periToneal, fallopIan, or oVarian cancEr (CREATIVE) trial is a prospective, multi-centre, phase IA dose-escalation study. Six cohorts of dose levels are planned, and six to 36 patients are expected to be enrolled in this trial. Major inclusion criteria include ≥ 19 years with histologically confirmed epithelial ovarian cancer, fallopian tube carcinoma, or primary peritoneal cancer. Also, patients who showed disease progression during platinum-based chemotherapy or disease progression within 24 weeks from completion of platinum-based chemotherapy will be included, and prior chemotherapy lines of more than five will be excluded. The primary endpoint of this study is to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of PHI-101. DISCUSSION: PHI-101 is the first orally available Chk2 inhibitor, expected to show effectiveness in treating recurrent ovarian cancer. Through this CREATIVE trial, DLT and MTD of this new targeted therapy can be confirmed to find the recommended dose for the phase II clinical trial. This study may contribute to developing a new combination regimen for the treatment of ovarian cancer. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04678102 .