Combined hepatocellular carcinoma-cholangiocarcinoma with a predominant HCC component: better survival and MRI-based prediction.

OBJECTIVES: To determine the optimal cutoff value of HCC% for predicting the outcome of patients with combined hepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA) and to investigate how preoperative MR features based on the Liver Imaging Reporting and Data System (LI-RADS ver. 2018) are related to the HCC% in cHCC-CCA. METHODS: The study enrolled 107 patients pathologically confirmed to have single cHCC-CCA according to the 2019 WHO classification. A receiver operating characteristic (ROC) curve was used to find the optimal cutoff value of HCC% based on overall survival (OS). The preoperative MR imaging features and clinicopathological findings were retrospectively evaluated and compared between the high HCC% and low HCC% groups. RESULTS: In total, 107 patients (mean age, males vs. females: 56.6 ± 10.7 years vs. 54.2 ± 12.8 years) were evaluated. Analysis of the relationship between HCC% and OS by ROC curve suggested that the optimal cutoff value was 65%, by which 51 (47.7%) patients were assigned to the high HCC% group. LI-RADS categorization (OR = 3.657, p = 0.006 vs. OR = 4.075, p = 0.004) and serum carcinoembryonic antigen (CEA) >5 ng/mL (OR = 0.348, p = 0.089 vs. OR = 0.298, p = 0.040) were significant predictors for HCC% in cHCC-CCA in both univariate and multivariate analysis. CONCLUSIONS: cHCC-CCA patients with HCC components higher than 65% tend to exhibit better overall survival, and MRI-based LI-RADS categorization and serum CEA level are valuable for identifying HCC% in cHCC-CCA preoperatively. KEY POINTS: • cHCC-CCA patients with HCC components higher than 65% tend to exhibit better overall survival. • MRI-based LI-RADS categorization and serum CEA level were significant predictors for HCC% in cHCC-CCA in both univariate and multivariate analyses and valuable for identifying HCC% in cHCC-CCA preoperatively.

SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

Evolution of the Cdk-activator Speedy/RINGO in vertebrates.

Successful completion of the cell cycle relies on the precise activation and inactivation of cyclin-dependent kinases (Cdks) whose activity is mainly regulated by binding to cyclins. Recently, a new family of Cdk regulators termed Speedy/RINGO has been discovered, which can bind and activate Cdks but shares no apparent amino acid sequence homology with cyclins. All Speedy proteins share a conserved domain of approximately 140 amino acids called "Speedy Box", which is essential for Cdk binding. Speedy/RINGO proteins display an important role in oocyte maturation in Xenopus. Interestingly, a common feature of all Speedy genes is their predominant expression in testis suggesting that meiotic functions may be the most important physiological feature of Speedy genes. Speedy homologs have been reported in mammals and can be traced back to the most primitive clade of chordates (Ciona intestinalis). Here, we investigated the evolution of the Speedy genes and have identified a number of new Speedy/RINGO proteins. Through extensive analysis of numerous species, we discovered diverse evolutionary histories: the number of Speedy genes varies considerably among species, with evidence of substantial gains and losses. Despite the interspecies variation, Speedy is conserved among most species examined. Our results provide a complete picture of the Speedy gene family and its evolution.

Crosstalk Between Metabolism and Immune Activity Reveals Four Subtypes With Therapeutic Implications in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by metabolic dysregulation and distinct immunological signatures. The interplay between metabolic and immune processes in the tumor microenvironment (TME) causes the complexity and heterogeneity of immunotherapy responses observed during ccRCC treatment. Herein, we initially identified two distinct metabolic subtypes (C1 and C2 subtypes) and immune subtypes (I1 and I2 subtypes) based on the occurrence of differentially expressed metabolism-related prognostic genes and immune-related components. Notably, we observed that immune regulators with upregulated expression actively participated in multiple metabolic pathways. Therefore, we further delineated four immunometabolism-based ccRCC subtypes (M1, M2, M3, and M4 subtypes) according to the results of the above classification. Generally, we found that high metabolic activity could suppress immune infiltration. Immunometabolism subtype classification was associated with immunotherapy response, with patients possessing the immune-inflamed, metabolic-desert subtype (M3 subtype) that benefits the most from immunotherapy. Moreover, differences in the shifts in the immunometabolism subtype after immunotherapy were observed in the responder and non-responder groups, with patients from the responder group transferring to subtypes with immune-inflamed characteristics and less active metabolic activity (M3 or M4 subtype). Immunometabolism subtypes could also serve as biomarkers for predicting immunotherapy response. To decipher the genomic and epigenomic features of the four subtypes, we analyzed multiomics data, including miRNA expression, DNA methylation status, copy number variations occurrence, and somatic mutation profiles. Patients with the M2 subtype possessed the highest VHL gene mutation rates and were more likely to be sensitive to sunitinib therapy. Moreover, we developed non-invasive radiomic models to reveal the status of immune activity and metabolism. In addition, we constructed a radiomic prognostic score (PRS) for predicting ccRCC survival based on the seven radiomic features. PRS was further demonstrated to be closely linked to immunometabolism subtype classification, immune score, and tumor mutation burden. The prognostic value of the PRS and the association of the PRS with immune activity and metabolism were validated in our cohort. Overall, our study established four immunometabolism subtypes, thereby revealing the crosstalk between immune and metabolic activities and providing new insights into personal therapy selection.

Simple Death Risk Models to Predict In-hospital Outcomes in Acute Aortic Dissection in Emergency Department.

Objective: We sought to find a bedside prognosis prediction model based on clinical and image parameters to determine the in-hospital outcomes of acute aortic dissection (AAD) in the emergency department. Methods: Patients who presented with AAD from January 2010 to December 2019 were retrospectively recruited in our derivation cohort. Then we prospectively collected patients with AAD from January 2020 to December 2021 as the validation cohort. We collected the demographics, medical history, treatment options, and in-hospital outcomes. All enrolled patients underwent computed tomography angiography. The image data were systematically reviewed for anatomic criteria in a retrospective fashion by three professional radiologists. A series of radiological parameters, including the extent of dissection, the site of the intimal tear, entry tear diameter, aortic diameter at each level, maximum false lumen diameter, and presence of pericardial effusion were collected. Results: Of the 449 patients in the derivation cohort, 345 (76.8%) were male, the mean age was 61 years, and 298 (66.4%) had a history of hypertension. Surgical repair was performed in 327 (72.8%) cases in the derivation cohort, and the overall crude in-hospital mortality of AAD was 10.9%. Multivariate logistic regression analysis showed that predictors of in-hospital mortality in AAD included age, Marfan syndrome, type A aortic dissection, surgical repair, and maximum false lumen diameter. A final prognostic model incorporating these five predictors showed good calibration and discrimination in the derivation and validation cohorts. As for type A aortic dissection, 3-level type A aortic dissection clinical prognosis score (3ADPS) including 5 clinical and image variables scored from -2 to 5 was established: (1) moderate risk of death if 3ADPS is <0; (2) high risk of death if 3ADPS is 1-2; (3) very high risk of death if 3ADPS is more than 3. The area under the receiver operator characteristic curves in the validation cohorts was 0.833 (95% CI, 0.700-0.967). Conclusion: Age, Marfan syndrome, type A aortic dissection, surgical repair, and maximum false lumen diameter can significantly affect the in-hospital outcomes of AAD. And 3ADPS contributes to the prediction of in-hospital prognosis of type A aortic dissection rapidly and effectively. As multivariable risk prediction tools, the risk models were readily available for emergency doctors to predict in-hospital mortality of patients with AAD in extreme clinical risk.

Ras1 and Ras2 play antagonistic roles in regulating cellular cAMP level, stationary-phase entry and stress response in Candida albicans.

The GTPase Ras1 activates the yeast-to-hypha transition in Candida albicans by activating cAMP synthesis. Here, we have characterized Ras2. Ras2 belongs to a group of atypical Ras proteins in some fungal species that share poor identity with other Ras GTPases with many variations in conserved motifs thought to be crucial for Ras-associated activities. We find that recombinant Ras2 is enzymatically as active as Ras1. However, only RAS1 can rescue the lethality of the Saccharomyces cerevisiae ras1 ras2 mutant, suggesting functional divergence of the two genes. ras2Delta is normal in hyphal growth, but deleting RAS2 in the ras1Delta background greatly aggravates the hyphal defect, indicating that Ras2 also has a role in hyphal development. Strikingly, while RAS1 deletion causes a approximately 20-fold decrease in cellular cAMP, further deletion of RAS2 restores it to approximately 30% of the wild-type level. Consistently, while the ras1Delta mutant enters the stationary phase prematurely, the double mutant does so normally. Moreover, ras1Delta cells exhibit increased resistance to H(2)O(2) and higher sensitivity to the heavy metal Co(2+), whereas ras2Delta cells show the opposite phenotypes. Together, our data reveal a novel regulatory mechanism by which two antagonizing Ras GTPases balance each other in regulating multiple cellular processes in C. albicans.

Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans.

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.

Cereblon Modulators Target ZBTB16 and Its Oncogenic Fusion Partners for Degradation via Distinct Structural Degrons.

There is a growing interest in using targeted protein degradation as a therapeutic modality in view of its potential to expand the druggable proteome. One avenue to using this modality is via molecular glue based Cereblon E3 Ligase Modulating Drug compounds. Here, we report the identification of the transcription factor ZBTB16 as a Cereblon neosubstrate. We also report two new Cereblon modulators, CC-3060 and CC-647, that promote ZBTB16 degradation. Unexpectedly, CC-3060 and CC-647 target ZBTB16 for degradation by primarily engaging distinct structural degrons on different zinc finger domains. The reciprocal fusion proteins, ZBTB16-RARα and RARα-ZBTB16, which cause a rare acute promyelocytic leukemia, contain these same structural degrons and can be targeted for proteasomal degradation with Cereblon modulator treatment. Thus, a targeted protein degradation approach via Cereblon modulators may represent a novel therapeutic strategy in acute promyelocytic leukemia where ZBTB16/RARA rearrangements are critical disease drivers.

Crystal structure of the SALL4-pomalidomide-cereblon-DDB1 complex.

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.

Role of Myocardial Extracellular Volume Fraction Measured with Magnetic Resonance Imaging in the Prediction of Left Ventricular Functional Outcome after Revascularization of Chronic Total Occlusion of Coronary Arteries.

Objective: The purpose of this study was to prospectively investigate the value of the myocardial extracellular volume fraction (ECV) in predicting myocardial functional outcome after revascularization of coronary chronic total occlusion (CTO). Materials and Methods: Thirty patients with CTO underwent cardiovascular magnetic resonance (CMR) before and 6 months after revascularization. Three baseline markers of functional outcome were evaluated in the dysfunctional segments assigned to the CTO vessels: ECV, transmural extent of infarction (TEI), and unenhanced rim thickness (RIM). At the global level, the ECV values of the whole myocardium with and without a hyperenhanced region (global and remote ECV) were respectively measured. Results: In per-segment analysis, ECV was superior to TEI and RIM in predicting functional recovery (area under receiver operating characteristic curve [AUC]: 0.86 vs. 0.75 and 0.73, all p values < 0.010), and it emerged as the only independent predictor of regional functional outcome (odds ratio [OR] = 0.83, 95% confidence interval [CI]: 0.77-0.89; p < 0.001) independent of collateral circulation. In per-patient analysis, global baseline ECV was indicative of ejection fraction (EF) at the follow-up examination (ß = -0.61, p < 0.001) and changes in EF (ß = -0.57, p = 0.001) in multivariate regression analysis. A patient with global baseline ECV less than 30.0% (AUC, 0.93; sensitivity 94%, specificity 80%) was more likely to demonstrate significant EF improvement (OR: 0.38; 95% CI: 0.17-0.85; p = 0.019). Conclusion: Extracellular volume fraction obtained by CMR may provide incremental value for the prediction of functional recovery both at the segmental and global levels in CTO patients, and may facilitate the identification of patients who can benefit from revascularization.

UBE2G1 governs the destruction of cereblon neomorphic substrates.

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.

Mitochondrial Aging Defects Emerge in Directly Reprogrammed Human Neurons due to Their Metabolic Profile.

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain, but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging, in contrast to iPSC differentiation. Here, we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression, impaired axonal mitochondrial morphologies, lower mitochondrial membrane potentials, reduced energy production, and increased oxidized proteins levels. In contrast, the fibroblasts from which iNs were generated show only mild age-dependent changes, consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed, OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging.

mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway.

Atherosclerosis (AS) is a chronic immuno­inflammatory disease accompanied by dyslipidemia. The authors previously demonstrated that sirtuin 1 (SIRT1) may prevent atherogenesis through influencing the liver X receptor/C­C chemokine receptor type 7/nuclear factor­κB (LXR­CCR7/NF­κB) signaling pathway. Previous studies have suggested a role for mammalian target of rapamycin (mTOR) signaling in the pathogenesis of cardiovascular diseases. The present study investigated the potential association between mTOR signaling and SIRT1­LXR­CCR7/NF­κB signaling (SIRT1 signaling) in AS pathogenesis. To induce foam cell formation, U937 cells were differentiated into macrophages by exposure to phorbol 12­myristate 13­acetate (PMA) for 24 h, followed by treatment with palmitate and oxidized low density lipoprotein for a further 24 h. Oil red O staining revealed a large accumulation of lipid droplets present in foam cells. Western blot analysis demonstrated increased protein levels of phosphorylated (p)­mTOR and its downstream factor p­ribosomal protein S6 kinase (p70S6K). Reverse transcription­quantitative polymerase chain reaction and western blot analyses additionally revealed decreased expression of SIRT1, LXRα and CCR7 and increased expression of NF­κB and its downstream factor tumor necrosis factor­α (TNF­α) in an atherogenetic condition induced by lysophosphatidic acid (LPA). In addition, abundant lipid droplets accumulated in U937­LPA­treated foam cells. Rapamycin, an mTOR inhibitor, suppressed the expression and activity of mTOR and p70S6K, however enhanced expression of SIRT1, LXRα, and CCR7. Conversely, rapamycin deceased TNF­α and NF­κB activity, the latter of which was further confirmed by immunofluorescence analysis demonstrating increased levels of NF­κB present in the cytoplasm compared with the nucleus. The findings of the present study suggest that mTOR signaling promotes foam cell formation and inhibits foam cell egress via suppression of SIRT1 signaling.

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration.

mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration.

How phosphoubiquitin activates Parkin.

A recent report, solving the structure of a Parkin-phosphoubiquitin complex, greatly advances the understanding of the Parkin activation mechanism.

Multimodal SPION-CREKA peptide based agents for molecular imaging of microthrombus in a rat myocardial ischemia-reperfusion model.

Microthrombosis plays a key role in many cardiovascular diseases. Although it is not difficult to localize thrombus within large or middle-sized vessels, the noninvasive diagnostic regimen for the detection of microthrombus remains scarce. Here we developed a nanoagent by conjucting superparamagnetic iron-oxide nanoparticle with fluorophore and a targeting element, CREKA, a peptide with special affinity for fibrin. In a rat model of myocardial ischemia-reperfusion (MI/R), the multimodal nanoagents were readily and selectively accumulated within microthrombosis, which was detectable by both magnetic resonance and optical imaging modalities. The fibrin-targeted nanoagent could be expected to have utility not only in molecular imaging of fibrin, understanding the mechanisms of microcirculation disorders, but also in targeted therapy with fibrinolytic agents.

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism.

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin's in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.

Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation.

The growing tips of Candida albicans hyphae are sites of polarized exocytosis. Mammalian septins have been implicated in regulating exocytosis and C. albicans septins are known to localize at hyphal tips, although their function here is unknown. Here, we report that C. albicans cells deleted of the exocyst subunit gene SEC3 can grow normal germ tubes, but are unable to maintain tip growth after assembly of the first septin ring, resulting in isotropic expansion of the tip. Deleting either of the septin genes CDC10 or CDC11 caused Sec3p mislocalization and surprisingly, also restored hyphal development in the sec3Delta mutant without rescuing the temperature sensitivity. Co-immunoprecipitation experiments detected association of the septin Cdc3p with the exocyst subunits Sec3p and Sec5p. Our results reveal that C. albicans hyphal development occurs through Sec3p-independent and dependent phases, and provide strong genetic and biochemical evidence for a role of septins in polarized exocytosis.