Structure-Dependent Water Responsiveness of Protein Block Copolymers.

Biological water-responsive (WR) materials are abundant in nature, and they are used as mechanical actuators for seed dispersal by many plants such as wheat awns and pinecones. WR biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural water evaporation. Recent work on WR silk proteins has shown that ß-sheet nanocrystalline domains with high stiffness correlate with the high WR actuation energy density, but the fundamental mechanisms to drive water responsiveness in proteins remain poorly understood. Here, we design, synthesize, and study protein block copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E), namely, CEC. We use these protein materials to create WR actuators with energy densities that outperform mammalian muscle. To elucidate the effect of structure on WR actuation, CEC was compared to a variant, CECL44A, in which a point mutation disrupts the α-helical structure of the C domain. Surprisingly, CECL44A outperformed CEC, showing higher energy density and less susceptibility to degradation after repeated cycling. We show that CECL44A exhibits a higher degree of intermolecular interactions and is stiffer than CEC at high relative humidity (RH), allowing for less energy dissipation during water responsiveness. These results suggest that strong intermolecular interactions and the resulting, relatively steady protein structure are important for water responsiveness.

"The Prognostic Role of Aspartate Transaminase to Platelet Ratio Index (APRI) on Outcomes Following Non-emergent Minor Hepatectomy".

INTRODUCTION: Fibrosis and cirrhosis are associated with worse outcomes after hepatectomy. Aspartate transaminase to platelet ratio index (APRI) is associated with fibrosis and cirrhosis in hepatitis C patients. However, APRI has not been studied to predict outcomes after hepatectomy in patients without viral hepatitis. METHODS: We reviewed the ACS-NSQIP dataset to identify patients who underwent a minor hepatectomy between 2014 and 2021. We excluded patients with viral hepatitis or ascites as well as patients who underwent emergent operations or biliary reconstruction. APRI was calculated using the following equation: (AST/40)/(platelet count) × 100. APRI ≥0.7 was used to identify significant fibrosis. Univariable analysis was performed to identify factors associated with APRI ≥0.7, transfusion, serious morbidity, overall morbidity, and 30-day mortality. Multivariable logistic regression was performed to identify adjusted predictors of these outcomes. RESULTS: Of the 18,069 patients who met inclusion criteria, 1630 (9.0%) patients had an APRI ≥0.7. A perioperative blood transfusion was administered to 2139 (11.8%). Overall morbidity, serious morbidity, and mortality were experienced by 3162 (17.5%), 2475 (13.7%), and 131 (.7%) patients, respectively. APRI ≥0.7 was an independent predictor of transfusion (adjusted OR: 1.48 [1.26-1.74], P < .001), overall morbidity (1.17 [1.02-1.33], P = .022), and mortality (1.97 [1.22-3.06], P = .004). Transfusion was an independent predictor of overall morbidity (3.31 [2.99-3.65], P < .001), serious morbidity (3.70 [3.33-4.11], P < .001), and mortality (5.73 [4.01-8.14], P < .001). CONCLUSIONS: APRI ≥0.7 is associated with perioperative transfusion, overall morbidity, and 30-day mortality. APRI may serve as a noninvasive tool to risk stratify patients prior to elective minor hepatectomy.

Coiled-Coil Protein Hydrogels Engineered with Minimized Fiber Diameters for Sustained Release of Doxorubicin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks expressed protein targets, making therapy development challenging. Hydrogels offer a promising new route in this regard by improving the chemotherapeutic efficacy through increased solubility and sustained release. Moreover, subcutaneous hydrogel administration reduces patient burden by requiring less therapy and shorter treatment times. We recently established the design principles for the supramolecular assembly of single-domain coiled-coils into hydrogels. Using a modified computational design algorithm, we designed Q8, a hydrogel with rapid assembly for faster therapeutic hydrogel preparation. Q8 encapsulates and releases doxorubicin (Dox), enabling localized sustained release via subcutaneous injection. Remarkably, a single subcutaneous injection of Dox-laden Q8 (Q8•Dox) significantly suppresses tumors within just 1 week. This work showcases the bottom-up engineering of a fully protein-based drug delivery vehicle for improved TBNC treatment via noninvasive localized therapy.

Engineered coiled-coil HIF1α protein domain mimic.

The development of targeted anti-cancer therapeutics offers the potential for increased efficacy of drugs and diagnostics. Utilizing modalities agnostic to tumor type, such as the hypoxic tumor microenvironment (TME), may assist in the development of universal tumor targeting agents. The hypoxia-inducible factor (HIF), in particular HIF1, plays a key role in tumor adaptation to hypoxia, and inhibiting its interaction with p300 has been shown to provide therapeutic potential. Using a multivalent assembled protein (MAP) approach based on the self-assembly of the cartilage oligomeric matrix protein coiled-coil (COMPcc) domain fused to the critical residues of the C-terminal transactivation domain (C-TAD) of the α subunit of HIF1 (HIF1α), we generate HIF1α-MAP (H-MAP). The resulting H-MAP demonstrates picomolar binding affinity to p300, the ability to downregulate hypoxia-inducible genes, and in vivo tumor targeting capability.

Design of Coiled-Coil Protein Nanostructures for Therapeutics and Drug Delivery.

Coiled-coil protein motifs have become widely employed in the design of biomaterials. Some of these designs have been studied for use in drug delivery due to the unique ability of coiled-coils to impart stability, oligomerization, and supramolecular assembly. To leverage these properties and improve drug delivery, release, and targeting, a variety of nano- to mesoscale architectures have been adopted. Coiled-coil drug delivery and therapeutics have been developed by using the coiled-coil alone, designing for higher-order assemblies such as fibers and hydrogels, and combining coiled-coil proteins with other biocompatible structures such as lipids and polymers. We review the recent development of these structures and the design criteria used to generate functional proteins of varying sizes and morphologies.

Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Computational Design of Phosphotriesterase Improves V-Agent Degradation Efficiency.

Organophosphates (OPs) are a class of neurotoxic acetylcholinesterase inhibitors including widely used pesticides as well as nerve agents such as VX and VR. Current treatment of these toxins relies on reactivating acetylcholinesterase, which remains ineffective. Enzymatic scavengers are of interest for their ability to degrade OPs systemically before they reach their target. Here we describe a library of computationally designed variants of phosphotriesterase (PTE), an enzyme that is known to break down OPs. The mutations G208D, F104A, K77A, A80V, H254G, and I274N broadly improve catalytic efficiency of VX and VR hydrolysis without impacting the structure of the enzyme. The mutation I106 A improves catalysis of VR and L271E abolishes activity, likely due to disruptions of PTE's structure. This study elucidates the importance of these residues and contributes to the design of enzymatic OP scavengers with improved efficiency.

Single-cell profile reveals the landscape of cardiac immunity and identifies a cardio-protective Ym-1hi neutrophil in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a major hindrance to the success of cardiac reperfusion therapy. Although increased neutrophil infiltration is a hallmark of MIRI, the subtypes and alterations of neutrophils in this process remain unclear. Here, we performed single-cell sequencing of cardiac CD45+ cells isolated from the murine myocardium subjected to MIRI at six-time points. We identified diverse types of infiltrating immune cells and their dynamic changes during MIRI. Cardiac neutrophils showed the most immediate response and largest changes and featured with functionally heterogeneous subpopulations, including Ccl3hi Neu and Ym-1hi Neu, which were increased at 6 h and 1 d after reperfusion, respectively. Ym-1hi Neu selectively expressed genes with protective effects and was, therefore, identified as a novel specific type of cardiac cell in the injured heart. Further analysis indicated that neutrophils and their subtypes orchestrated subsequent immune responses in the cardiac tissues, especially instructing the response of macrophages. The abundance of Ym-1hi Neu was closely correlated with the therapeutic efficacy of MIRI when neutrophils were specifically targeted by anti-Lymphocyte antigen 6 complex locus G6D (Ly6G) or anti-Intercellular cell adhesion molecule-1 (ICAM-1) neutralizing antibodies. In addition, a neutrophil subtype with the same phenotype as Ym-1hi Neu was detected in clinical samples and correlated with prognosis. Ym-1 inhibition exacerbated myocardial injury, whereas Ym-1 supplementation significantly ameliorated injury in MIRI mice, which was attributed to the tilt of Ym-1 on the polarization of macrophages toward the repair phenotype in myocardial tissue. Overall, our findings reveal the anti-inflammatory phenotype of Ym-1hi Neu and highlight its critical role in myocardial protection during the early stages of MIRI.

One-Year Clinical Outcomes of Subcutaneous Infliximab Maintenance Therapy Compared With Intravenous Infliximab Maintenance Therapy in Patients With Inflammatory Bowel Disease: A Prospective Cohort Study.

BACKGROUND: Although the pharmacokinetic profile of subcutaneous (SC) infliximab (IFX) is superior to conventional intravenous (IV) IFX, long-term efficacy and safety of SC IFX in patients with inflammatory bowel disease (IBD) have not been reported yet. This study aimed to evaluate long-term clinical outcomes of IBD patients treated with SC IFX compared with those of IBD patients treated with IV IFX during maintenance therapy. METHODS: This prospective cohort study enrolled 61 IBD patients in clinical remission who received scheduled IFX maintenance therapy. Of them, 38 patients were switched to SC IFX, while 23 patients continued IV IFX with dose optimization. Enrolled patients were followed up for 1 year. The primary outcome was durable remission defined as clinical remission (Crohn's disease, Harvey-Bradshaw index <5; ulcerative colitis, partial Mayo score <2) and biochemical remission (C-reactive protein <0.5 mg/dL) with IFX trough level ≥3 µg/mL throughout the follow-up period. RESULTS: One-year clinical remission, 1-year biochemical remission, and mucosal healing did not differ between the IV and SC IFX groups (n = 20 of 23 vs 33 of 38; P = 1.000; n = 22 of 23 vs 34 of 38; P = .641; and n = 10 of 18 vs 17 of 25; P = .414, respectively). During follow-up, the number of patients with IFX trough level <3 µg/mL was significantly lower in the SC IFX group (n = 0 of 38, 0%) than in the IV IFX group (n = 10 of 23, 43%) (P < .001). The SC IFX group showed higher 1-year durable remission than the IV IFX group (n = 31 of 38, 82% vs n = 11 of 23, 48%; P = .013). The incidence of IFX-related adverse events did not differ significantly between both groups (26% vs 39%; P = .446). CONCLUSION: The SC IFX switch induced a higher 1-year durable remission rate than continuing IV IFX in patients with IBD during scheduled maintenance therapy, showing similar safety.

Long-term efficacy and safety of subcutaneous infliximab in patients with inflammatory bowel diseases have not been reported yet. Switching from intravenous to subcutaneous infliximab showed higher 1-year durable remission than continuing intravenous infliximab during scheduled maintenance therapy, with similar safety.

Computational Prediction of Coiled-Coil Protein Gelation Dynamics and Structure.

Protein hydrogels represent an important and growing biomaterial for a multitude of applications, including diagnostics and drug delivery. We have previously explored the ability to engineer the thermoresponsive supramolecular assembly of coiled-coil proteins into hydrogels with varying gelation properties, where we have defined important parameters in the coiled-coil hydrogel design. Using Rosetta energy scores and Poisson-Boltzmann electrostatic energies, we iterate a computational design strategy to predict the gelation of coiled-coil proteins while simultaneously exploring five new coiled-coil protein hydrogel sequences. Provided this library, we explore the impact of in silico energies on structure and gelation kinetics, where we also reveal a range of blue autofluorescence that enables hydrogel disassembly and recovery. As a result of this library, we identify the new coiled-coil hydrogel sequence, Q5, capable of gelation within 24 h at 4 °C, a more than 2-fold increase over that of our previous iteration Q2. The fast gelation time of Q5 enables the assessment of structural transition in real time using small-angle X-ray scattering (SAXS) that is correlated to coarse-grained and atomistic molecular dynamics simulations revealing the supramolecular assembling behavior of coiled-coils toward nanofiber assembly and gelation. This work represents the first system of hydrogels with predictable self-assembly, autofluorescent capability, and a molecular model of coiled-coil fiber formation.

Thiazol-4(5H)-one analogs as potent tyrosinase inhibitors: Synthesis, tyrosinase inhibition, antimelanogenic effect, antioxidant activity, and in silico docking simulation.

As the ß-phenyl-α,ß-unsaturated carbonyl (PUSC) structure was previously identified to play a key role in tyrosinase inhibition, 14 analogs with a PUSC structure built on a thiazol-4(5H)-one scaffold were synthesized using Knoevenagel condensation to serve as potential tyrosinase inhibitors. Through mushroom tyrosinase inhibition experiments, two analogs 9 and 11 were identified as potent tyrosinase inhibitors, with 11 exhibiting an IC50 value of 0.4 ± 0.01 µM, which indicates its 26-fold greater potency than kojic acid. Kinetic studies using Lineweaver-Burk plots revealed that 9 and 11 are competitive and mixed-type inhibitors, respectively; these kinetic results were supported by docking simulations. According to the B16F10 cell-based experiments, 9 and 11 inhibited melanogenesis more effectively than kojic acid due to their potent cellular tyrosinase inhibitory activity. In addition, analogs 9 and 11 exhibited moderate-to-strong antioxidant capacity, scavenging ABTS+, DPPH, and ROS radicals. In particular, analog 12 with a catechol moiety exhibited very strong ROS-scavenging activity, similar to Trolox. These results suggest that analogs 9 and 11, which exhibit potent tyrosinase inhibitory activity in mushroom and mammalian cells and anti-melanogenic effects in B16F10 cells, are promising antibrowning agents for crops and skin lightening agents for hyperpigmentation-related diseases.

Protein-Engineered Fibers For Drug Encapsulation Traceable via 19F Magnetic Resonance.

Theranostic materials research is experiencing rapid growth driven by the interest in integrating both therapeutic and diagnostic modalities. These materials offer the unique capability to not only provide treatment but also track the progression of a disease. However, to create an ideal theranostic biomaterial without compromising drug encapsulation, diagnostic imaging must be optimized for improved sensitivity and spatial localization. Herein, we create a protein-engineered fluorinated coiled-coil fiber, Q2TFL, capable of improved sensitivity to 19F magnetic resonance spectroscopy (MRS) detection. Leveraging residue-specific noncanonical amino acid incorporation of trifluoroleucine (TFL) into the coiled-coil, Q2, which self-assembles into nanofibers, we generate Q2TFL. We demonstrate that fluorination results in a greater increase in thermostability and 19F magnetic resonance detection compared to the nonfluorinated parent, Q2. Q2TFL also exhibits linear ratiometric 19F MRS thermoresponsiveness, allowing it to act as a temperature probe. Furthermore, we explore the ability of Q2TFL to encapsulate the anti-inflammatory small molecule, curcumin (CCM), and its impact on the coiled-coil structure. Q2TFL also provides hyposignal contrast in 1H MRI, echogenic signal with high-frequency ultrasound and sensitive detection by 19F MRS in vivo illustrating fluorination of coiled-coils for supramolecular assembly and their use with 1H MRI, 19F MRS and high frequency ultrasound as multimodal theranostic agents.

Strategy for dealing with unfamiliar and thick vessels during microvascular decompression: A first case report of hemifacial spasm caused by a persistent primitive trigeminal artery.

RATIONALE: A persistent primitive trigeminal artery (PPTA) is a rare embryonic cerebrovascular anomaly. Hemifacial spasm (HFS) refers to involuntary contractions of facial muscles caused by the compression of blood vessels against the root exit zone of the facial nerve. There have been no reported cases of PPTA causing neurovascular contact and HFS. Microvascular decompression surgery effectively treats HFS, but operating on strong PPTA vessels poses challenges. We aim to introduce a more efficient approach for overcomes these difficulties and facilitates surgery. PATIENT CONCERNS: A 44-year-old male patient without any underlying medical conditions presented to our hospital with involuntary movements of the left side of his face accompanied by numbness in the left maxilla (V2 area). DIAGNOSIS: Brain magnetic resonance imaging and magnetic resonance angiography showed that PPTA was in contact with the left facial nerve. INTERVENTIONS AND OUTCOMES: Following a retro-sigmoid craniotomy, we attempted to interpose the facial nerve and the PPTA as an offender vessel, but the decompression was not sufficient. However, after transposing the vessel using the proximal Teflon transposition with interposition technique, the strength of the involuntary movements was reduced. Following surgery, there was no more lateral spreading response, and the patient symptoms improved. LESSIONS: In cases where the vessel causing HFS is particularly strong and thick, the proximal Teflon transposition with interposition technique for transposition may be advantageous. This method could simplify and enhance the efficacy of microvascular decompression, without compromising the quality of surgical outcomes.

Vactosertib, a novel TGF-ß1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma.

Functional blockade of the transforming growth factor-beta (TGF-ß) signaling pathway improves the efficacy of cytotoxic and immunotherapies. We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF-ß type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma patients who had received ≥2 lines of chemoimmunotherapy. Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, while pomalidomide alone historically achieved 20% PFS-6. Following treatment, the immunosuppressive marker PD-1 expression was reduced on patient CD8+ T-cells. Following ex vivo treatment, vactosertib decreased PD-1 expression on patient CD138+ cells, reduced PD-L1/PD-L2 on patient CD138+ cells and enhanced the anti-myeloma activity of autologous T-cells. Taken together, vactosertib is a safe immunotherapy that modulates the T-cell immunophenotype to reinvigorate T-cell fitness. Multiple myeloma (MM) is a genetically heterogeneous hematologic malignancy characterized by the excessive proliferation of clonal plasma cells (1, 2). MM remains mostly incurable but a small group of patients can achieve long-term remission (3). Treatment of MM presents unique challenges due to the complex molecular pathophysiology and genetic heterogeneity (4, 5). Given that MM is the second most common blood cancer characterized by cycles of remission and relapse, the development of new therapeutic modalities is crucial (6, 7). The prognosis for MM patients has improved substantially over the past two decades with the development of more effective therapeutics, e.g., proteasome inhibitors, and regimens that demonstrate greater anti-tumor activity (8-10). The management of RRMM represents a vital aspect of the overall care for patients with disease and a critical area of ongoing scientific and clinical research (10-12).

Fermented Kamut Sprout Extract Decreases Cell Cytotoxicity and Increases the Anti-Oxidant and Anti-Inflammation Effect.

Kamut sprouts (KaS) contain several biologically active compounds. In this study, solid-state fermentation using Saccharomyces cerevisiae and Latilactobacillus sakei was used to ferment KaS (fKaS-ex) for 6 days. The fKaS-ex showed a 26.3 mg/g dried weight (dw) and 46.88 mg/g dw of polyphenol and the ß-glucan contents, respectively. In the Raw264.7 and HaCaT cell lines, the non-fermented KaS (nfKaS-ex) decreased cell viability from 85.3% to 62.1% at concentrations of 0.63 and 2.5 mg/mL, respectively. Similarly, the fKaS-ex decreased cell viability, but showed more than 100% even at 1.25 and 5.0 mg/mL concentrations, respectively. The anti-inflammatory effect of fKaS-ex also increased. At 600 µg/mL, the fKaS-ex exhibited a significantly higher ability to reduce cytotoxicity by suppressing COX-2 and IL-6 mRNA expressions as well as that for IL-1ß mRNA. In summary, fKaS-ex exhibited significantly lower cytotoxicity and increased anti-oxidant and anti-inflammatory properties, indicating that fKaS-ex is beneficial for use in food and other industries.

Development of a method for analysis and risk assessment of residual pesticides in ginseng using liquid and gas chromatography-tandem mass spectrometry.

In this study, we developed a method for detecting 335 pesticides in ginseng using liquid chromatography quadrupole mass spectrometry (LC-MS/MS) and gas chromatography quadrupole mass spectrometry (GC-MS/MS). Additionally, the linearity, sensitivity, selectivity, accuracy, and precision of the method was validated. The limits of detection (LOD) and limits of quantification (LOQ) for the instrument used in these experiments was 0.1-5.8 µg/kg and 0.3-17.5 µg/kg, respectively. The average recovery was 71.6-113.4%. From 2016 to 2019, 467 ginseng samples were analyzed, of which 304 samples detected pesticide residues, but most of them were below the standard. It can be observed that the hazard quotient (HQ) of ginseng for detected pesticides was less than 1, thus implying that the risk was low. Hence, in this study, we developed a specific, reliable, and suitable method for a fast and simultaneous analysis of 335 pesticides in ginseng.

Discrimination between Korean and Chinese Kimchi using inductively coupled plasma-optical emission spectroscopy and mass spectrometry: A multivariate analysis of Kimchi.

Kimchi has been designated as one of the world's five healthiest foods, and it is a traditional Korean fermented food. The purpose of this study was to investigate whether the origin of Kimchi can be discriminated against by using inorganic elements to develop a more accurate method. The OPLS-DA showed that the R2 and Q2 values were 0.908 and 0.81, a high-quality model. We selected 24 elements (133Cs, 238U, 88Sr, K, 157Gd, Na, Mg, 139La, P, 141Pr, 72Ge, 146Nd, 147Sm, 153Eu, 55Mn, 165Ho, 163Dy, 166Er, Fe, 172Yb, 169Tm, 185Re, 175Lu, and 118Sn) with VIP 1 or higher in the OPLS-DA model. In ROC, the selected elements had an accuracy of 100%. The heatmap confirmed the contents of rare earth elements (REEs) in Korean and Chinese Kimchi, the accuracy of distinguishing classification in CDA is 100%. Such results will help to distinguish the origin of agricultural products through inorganic analysis.

Timosaponin AIII promotes non-small-cell lung cancer ferroptosis through targeting and facilitating HSP90 mediated GPX4 ubiquitination and degradation.

Timosaponin AIII (Tim-AIII), a steroid saponin, exhibits strong anticancer activity in a variety of cancers, especially breast cancer and liver cancer. However, the underlying mechanism of the effects of Tim-AIII-mediated anti-lung cancer effects remain obscure. In this study, we showed that Tim-AIII suppressed cell proliferation and migration, induced G2/M phase arrest and ultimately triggered cell death of non-small cell lung cancer (NSCLC) cell lines accompanied by the release of reactive oxygen species (ROS) and iron accumulation, malondialdehyde (MDA) production, and glutathione (GSH) depletion. Interestingly, we found that Tim-AIII-mediated cell death was reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). Meanwhile, the heat shock protein 90 (HSP90) was predicted and verified as the direct binding target of Tim-AIII by SwissTargetPrediction (STP) and surface plasmon resonance (SPR) assay. Further study showed that Tim-AIII promoted HSP90 expression and Tim-AIII induced cell death was blocked by the HSP90 inhibitor tanespimycin, indicating that HSP90 was the main target of Tim-AIII to further trigger intracellular events. Mechanical analysis revealed that the Tim-AIII-HSP90 complex further targeted and degraded glutathione peroxidase 4 (GPX4), and promoted the ubiquitination of GPX4, as shown by an immunoprecipitation, degradation and in vitro ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

The intra-tumoral heterogeneity in glioblastoma - a limitation for prognostic value of epigenetic markers?

OBJECTIVE: Epigenetic tumor features are getting into focus as prognostic markers in glioblastoma. Whether intra-tumoral heterogeneity in these epigenetic characteristics may influence prognostic value remains unclear. METHODS: Of 154 patients suffering from glioblastoma, 120 patients served as reference collective, while 34 patients were compiled as test collective. MGMT, p15, and p16 promoter methylation and miRNA expression levels (miRNA-21, miRNA-24, miRNA-26a, and miRNA-181d) were measured in each tumor specimen. Serving as a statistical baseline, epigenetic heterogeneity between tumors (inter-tumoral) was estimated within a triplet of three tumor specimens from three different reference patients. For estimation of epigenetic heterogeneity within a tumor (intra-tumoral), previous results were compared to three tumor specimens within one glioblastoma of patients of the test collective. Resulting levels of heterogeneity were then correlated with survival and validated by an external TCGA data set. RESULTS: Heterogeneity in MGMT promoter methylation occurred less likely in the test group compared to the reference group. No difference in heterogeneity was observed between test and reference group regarding p15 and p16 methylation. Intra-tumoral heterogeneity within the test group regarding miRNA-21, miRNA-24, miRNA-26a, and miRNA-181d expression was not distinguishable from inter-tumoral heterogeneity. A homogenously increased miRNA-21 expression was associated with reduced overall survival in the test collective. The findings could be validated by comparison with TCGA datasets. CONCLUSION: Heterogeneity of epigenetic characteristics in one glioblastoma may be of the same magnitude as heterogeneity between different patients. Not only the extent of epigenetic characteristics but also the extent of intra-tumoral heterogeneity may influence survival in glioblastoma.