Development of a RNA-protein complex based smart drug delivery system for 9-hydroxycamptothecin.

Camptothecin (CPT) is a monoterpenoid indole alkaloid with a wide spectrum of anticancer activity. However, its application is hindered by poor solubility, lack of targeting specificity, and severe side effects. Structural derivatization of CPT and the development of suitable drug delivery systems are potential strategies for addressing these issues. In this study, we discovered that the protein Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) from Homo sapiens catalyzes CPT to yield 9-hydroxycamptothecin (9-HCPT), which exhibits increased water solubility and cytotoxicity. We then created a RNA-protein complex based drug delivery system with enzyme and pH responsiveness and improved the targeting and stability of the nanomedicine through protein module assembly. The subcellular localization of nanoparticles can be visualized using fluorescent RNA probes. Our results not only identified the protein CYP1A1 responsible for the structural derivatization of CPT to synthesize 9-HCPT but also offered potential strategies for enhancing the utilization of silk-based drug delivery systems in tumor therapy.

Semisynthesis and antitumor activity of endertiin B and related triterpenoids from Ganoderma lucidum.

Ganoderma lucidum, a fungus used in traditional Chinese medicine, is known for its medicinal value attributed to its active components called Ganoderma triterpenoids (GTs). However, the limited isolation rate of these GTs has hindered their potential as promising drug candidates. Therefore, it is imperative to achieve large-scale preparation of GTs. In this study, four GTs were effectively synthesised from lanosterol. The antitumor activity of these GTs was evaluated in vivo. Endertiin B exhibited potent inhibitory activity against breast cancer cells (9.85 ± 0.91 µM and 12.12 ± 0.95 µM). Further investigations demonstrated that endertiin B significantly upregulated p21 and p27 and downregulated cyclinD1 expression, arresting the cell cycle at the G0/G1 phase and inducing apoptosis by decreasing BCL-2 and increasing BAX and BAK levels. Additionally, endertiin B was found to reduce the expression of proteins associated with the PI3K-AKT signaling pathway. To summarize, endertiin B effectively inhibited cell proliferation by blocking the cell cycle and inducing apoptosis through the PI3K-AKT pathway.

Short-Term Outcomes of Transcatheter Aortic Valve Replacement in Low-Risk Patients With Pure Severe Aortic Regurgitation.

Transcatheter aortic valve replacement (TAVR) has emerged as an alternative treatment for patients with pure severe aortic regurgitation (PSAR) who are contraindicated for surgery or have a high surgical risk. However, the therapeutic efficacy and safety of TAVR in low Society of Thoracic Surgeons (STS) score risk patients remain to be clarified. This study aimed to explore the feasibility of TAVR treatment in different STS-risk patients and to compare the adverse events between the groups. In this study, patients with PSAR who underwent TAVR at Zhongshan Hospital, Fudan University, China, during the inclusion period were included and categorized into 3 groups based on STS scores. The baseline data, imaging results, and follow-up data of the patients were documented. Therefore, of 75 TAVR patients, 38 (50.7%) were categorized as low risk (STS <4), and 37 (49.3%) patients were categorized as intermediate and high risk (STS ≥4). Compared with patients at intermediate and high risk, those in the low-risk group were younger, had a lower body mass index, had a lower prevalence of hypertension, chronic obstructive pulmonary disease, and previous percutaneous coronary intervention, and had better cardiac function (p all <0.05). In the hospital and at the 1-month follow-up, the degree of aortic regurgitation and cardiac function were significantly improved. No significant difference was found between the 2 groups in the hospital or during the 30-day follow-up. In conclusion, TAVR for PSAR in low-STS-risk patients is safe and efficient during 30 days of follow-up compared with intermediate- and high-STS-risk groups. TAVR for PSAR should not be limited to inoperable or STS-defined high-risk patients. Long-term follow-up is needed for further investigation.

Correction to "Diterpenoid Tanshinones Can Inhibit Lung Cancer Progression by Improving the Tumor Microenvironment and Downregulation of NF-κB Expression".

[This corrects the article DOI: 10.1021/acsomega.3c09667.].

Combined anti-PD-1, HDAC inhibitor and anti-VEGF for MSS/pMMR colorectal cancer: a randomized phase 2 trial.

Epigenetic modifications of chromatin, including histone acetylation, and tumor angiogenesis play pivotal roles in creating an immunosuppressive tumor microenvironment. In the randomized phase 2 CAPability-01 trial, we investigated the potential efficacy of combining the programmed cell death protein-1 (PD-1) monoclonal antibody sintilimab with the histone deacetylase inhibitor (HDACi) chidamide with or without the anti-vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab in patients with unresectable chemotherapy-refractory locally advanced or metastatic microsatellite stable/proficient mismatch repair (MSS/pMMR) colorectal cancer. Forty-eight patients were randomly assigned to either the doublet arm (sintilimab and chidamide, n = 23) or the triplet arm (sintilimab, chidamide and bevacizumab, n = 25). The primary endpoint of progression-free survival (PFS) rate at 18 weeks (18wPFS rate) was met with a rate of 43.8% (21 of 48) for the entire study population. Secondary endpoint results include a median PFS of 3.7 months, an overall response rate of 29.2% (14 of 48), a disease control rate of 56.3% (27 of 48) and a median duration of response of 12.0 months. The secondary endpoint of median overall survival time was not mature. The triplet arm exhibited significantly improved outcomes compared to the doublet arm, with a greater 18wPFS rate (64.0% versus 21.7%, P = 0.003), higher overall response rate (44.0% versus 13.0%, P = 0.027) and longer median PFS rate (7.3 months versus 1.5 months, P = 0.006). The most common treatment-emergent adverse events observed in both the triplet and doublet arms included proteinuria, thrombocytopenia, neutropenia, anemia, leukopenia and diarrhea. There were two treatment-related fatalities (hepatic failure and pneumonitis). Analysis of bulk RNA sequencing data from the patients suggested that the triplet combination enhanced CD8+ T cell infiltration, resulting in a more immunologically active tumor microenvironment. Our study suggests that the combination of a PD-1 antibody, an HDACi, and a VEGF antibody could be a promising treatment regimen for patients with MSS/pMMR advanced colorectal cancer. ClinicalTrials.gov registration: NCT04724239 .

Systemically functional characterization of regiospecific flavonoid O-methyltransferases from Glycine max.

Plants produce diverse flavonoids for defense and stress resistance, most of which have health benefits and are widely used as food additives and medicines. Methylation of the free hydroxyl groups of flavonoids, catalyzed by S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs), significantly affects their physicochemical properties and bioactivities. Soybeans (Glycine max) contain a rich pool of O-methylated flavonoids. However, the OMTs responsible for flavonoid methylation in G. max remain largely unknown. We screened the G. max genome and obtained 22 putative OMT-encoding genes that share a broad spectrum of amino acid identities (25-96%); among them, 19 OMTs were successfully cloned and heterologously expressed in Escherichia coli. We used the flavonoids containing the free 3, 5, 7, 8, 3', 4' hydroxyl group, such as flavones (luteolin and 7, 8-dihydroxyflavone), flavonols (kaempferol and quercetin), flavanones (naringenin and eriodictyol), isoflavonoids (daidzein and glycetein), and caffeic acid as substrates, and 15 OMTs were proven to catalyze at least one substrate. The methylation activities of these GmOMTs covered the 3, 7, 8, 3', 4'- hydroxyl of flavonoids and 7, 4'- hydroxyl of isoflavonoids. The systematic characterization of G. max flavonoid OMTs provides insights into the biosynthesis of methylated flavonoids in soybeans and OMT bioparts for the production of methylated flavonoids via synthetic biology.

Biosynthesis of the highly oxygenated tetracyclic core skeleton of Taxol.

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9α-hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1ß-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5α-ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol.

Artificial intelligence-assisted system for the assessment of Forrest classification of peptic ulcer bleeding: a multicenter diagnostic study.

BACKGROUND: Inaccurate Forrest classification may significantly affect clinical outcomes, especially in high risk patients. Therefore, this study aimed to develop a real-time deep convolutional neural network (DCNN) system to assess the Forrest classification of peptic ulcer bleeding (PUB). METHODS: A training dataset (3868 endoscopic images) and an internal validation dataset (834 images) were retrospectively collected from the 900th Hospital, Fuzhou, China. In addition, 521 images collected from four other hospitals were used for external validation. Finally, 46 endoscopic videos were prospectively collected to assess the real-time diagnostic performance of the DCNN system, whose diagnostic performance was also prospectively compared with that of three senior and three junior endoscopists. RESULTS: The DCNN system had a satisfactory diagnostic performance in the assessment of Forrest classification, with an accuracy of 91.2% (95%CI 89.5%-92.6%) and a macro-average area under the receiver operating characteristic curve of 0.80 in the validation dataset. Moreover, the DCNN system could judge suspicious regions automatically using Forrest classification in real-time videos, with an accuracy of 92.0% (95%CI 80.8%-97.8%). The DCNN system showed more accurate and stable diagnostic performance than endoscopists in the prospective clinical comparison test. This system helped to slightly improve the diagnostic performance of senior endoscopists and considerably enhance that of junior endoscopists. CONCLUSION: The DCNN system for the assessment of the Forrest classification of PUB showed satisfactory diagnostic performance, which was slightly superior to that of senior endoscopists. It could therefore effectively assist junior endoscopists in making such diagnoses during gastroscopy.

Diterpenoid Tanshinones Can Inhibit Lung Cancer Progression by Improving the Tumor Microenvironment and Downregulation of NF-κB Expression.

Diterpenoid tanshinones (DTs) are a bioactive fraction extracted from Salvia miltiorrhiza. High-performance liquid chromatography analysis revealed the presence of four compounds, namely, tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone. In this study, we aimed to propose a possible mechanism for the anti-lung cancer effect of DT. To do so, we utilized a lung cancer nude mice model and a lung cancer cell line (PC9) to investigate the effect of DT on lung cancer. We employed immunohistochemistry, enzyme-linked immunosorbent assay, hematoxylin and eosin staining, and immunofluorescence to analyze the pharmacological role of DT in the inhibition of lung cancer growth. The results showed that DT inhibited tumor growth, induced apoptosis in the nude mice model, and reduced inflammatory cell infiltration. Additionally, DT inhibited PC9 lung cancer cells, growth, proliferation, and migration. The mechanism of action of DT involves not only directly inhibiting cell proliferation and migration but also improving the tumor microenvironment. DT significantly increased the expression of important intestinal gap junction proteins, such as zonula occludens 1 (ZO-1) and occludin I. This upregulation contributes to the reinforcement of the intestinal mucosal barrier, thereby reducing the paracellular transport of lipopolysaccharides (LPS) through the intestine. Consequently, the decreased LPS levels lead to the inhibition of NF-κB expression and downregulation of macrophage polarization, as indicated by the decreased expression of CD68. In conclusion, this study has confirmed that DT has anti-lung cancer properties by improving the inflammatory tumor microenvironment via regulating macrophage polarization and inhibiting LPS-associated immune response. These results provide new insights into the mechanism of DT action against lung cancer.

Modulator of TMB-associated immune infiltration (MOTIF) predicts immunotherapy response and guides combination therapy.

Patients with high tumor mutational burden (TMB) levels do not consistently respond to immune checkpoint inhibitors (ICIs), possibly because a high TMB level does not necessarily result in adequate infiltration of CD8+ T cells. Using bulk ribonucleic acid sequencing (RNA-seq) data from 9311 tumor samples across 30 cancer types, we developed a novel tool called the modulator of TMB-associated immune infiltration (MOTIF), which comprises genes that can determine the extent of CD8+ T cell infiltration prompted by a certain TMB level. We confirmed that MOTIF can accurately reflect the integrity and defects of the cancer-immunity cycle. By analyzing 84 human single-cell RNA-seq datasets from 32 types of solid tumors, we revealed that MOTIF can provide insights into the diverse roles of various cell types in the modulation of CD8+ T cell infiltration. Using pretreatment RNA-seq data from 13 ICI-treated cohorts, we validated the use of MOTIF in predicting CD8+ T cell infiltration and ICI efficacy. Among the components of MOTIF, we identified EMC3 as a negative regulator of CD8+ T cell infiltration, which was validated via in vivo studies. Additionally, MOTIF provided guidance for the potential combinations of programmed death 1 blockade with certain immunostimulatory drugs to facilitate CD8+ T cell infiltration and improve ICI efficacy.

Machine learning-based extrachromosomal DNA identification in large-scale cohorts reveals its clinical implications in cancer.

The clinical implications of extrachromosomal DNA (ecDNA) in cancer therapy remain largely elusive. Here, we present a comprehensive analysis of ecDNA amplification spectra and their association with clinical and molecular features in multiple cohorts comprising over 13,000 pan-cancer patients. Using our developed computational framework, GCAP, and validating it with multifaceted approaches, we reveal a consistent pan-cancer pattern of mutual exclusivity between ecDNA amplification and microsatellite instability (MSI). In addition, we establish the role of ecDNA amplification as a risk factor and refine genomic subtypes in a cohort from 1015 colorectal cancer patients. Importantly, our investigation incorporates data from four clinical trials focused on anti-PD-1 immunotherapy, demonstrating the pivotal role of ecDNA amplification as a biomarker for guiding checkpoint blockade immunotherapy in gastrointestinal cancer. This finding represents clinical evidence linking ecDNA amplification to the effectiveness of immunotherapeutic interventions. Overall, our study provides a proof-of-concept of identifying ecDNA amplification from cancer whole-exome sequencing (WES) data, highlighting the potential of ecDNA amplification as a valuable biomarker for facilitating personalized cancer treatment.

The anti-leukemia activity and mechanisms of shikonin: a mini review.

Leukemia encompasses a group of highly heterogeneous diseases that pose a serious threat to human health. The long-term outcome of patients with leukemia still needs to be improved and new effective therapeutic strategies continue to be an unmet clinical need. Shikonin (SHK) is a naphthoquinone derivative that shows multiple biological function includes anti-tumor, anti-inflammatory, and anti-allergic effects. Numerous studies have reported the anti-leukemia activity of SHK during the last 3 decades and there are studies showing that SHK is particularly effective towards various leukemia cells compared to solid tumors. In this review, we will discuss the anti-leukemia effect of SHK and summarize the underlying mechanisms. Therefore, SHK may be a promising agent to be developed as an anti-leukemia drug.

Residual circulating tumor DNA after adjuvant chemotherapy effectively predicts recurrence of stage II-III gastric cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for predicting relapse in multiple solid cancers. However, the predictive value of ctDNA for disease recurrence remains indefinite in locoregional gastric cancer (GC). Here, we aimed to evaluate the predictive value of ctDNA in this context. METHODS: From 2016 to 2019, 100 patients with stage II/III resectable GC were recruited in this prospective cohort study (NCT02887612). Primary tumors were collected during surgical resection, and plasma samples were collected perioperatively and within 3 months after adjuvant chemotherapy (ACT). Somatic variants were captured via a targeted sequencing panel of 425 cancer-related genes. The plasma was defined as ctDNA-positive only if one or more variants detected in the plasma were presented in at least 2% of the primary tumors. RESULTS: Compared with ctDNA-negative patients, patients with positive postoperative ctDNA had moderately higher risk of recurrence [hazard ratio (HR) = 2.74, 95% confidence interval (CI) = 1.37-5.48; P = 0.003], while patients with positive post-ACT ctDNA showed remarkably higher risk (HR = 14.99, 95% CI = 3.08-72.96; P < 0.001). Multivariate analyses indicated that both postoperative and post-ACT ctDNA positivity were independent predictors of recurrence-free survival (RFS). Moreover, post-ACT ctDNA achieved better predictive performance (sensitivity, 77.8%; specificity, 90.6%) than both postoperative ctDNA and serial cancer antigen. A comprehensive model incorporating ctDNA for recurrence risk prediction showed a higher C-index (0.78; 95% CI = 0.71-0.84) than the model without ctDNA (0.71; 95% CI = 0.64-0.79; P = 0.009). CONCLUSIONS: Residual ctDNA after ACT effectively predicts high recurrence risk in stage II/III GC, and the combination of tissue-based and circulating tumor features could achieve better risk prediction.

The liver microenvironment orchestrates FGL1-mediated immune escape and progression of metastatic colorectal cancer.

Colorectal cancer (CRC) patients with liver metastases usually obtain less benefit from immunotherapy, and the underlying mechanisms remain understudied. Here, we identify that fibrinogen-like protein 1 (FGL1), secreted from cancer cells and hepatocytes, facilitates the progression of CRC in an intraportal injection model by reducing the infiltration of T cells. Mechanistically, tumor-associated macrophages (TAMs) activate NF-ĸB by secreting TNFα/IL-1ß in the liver microenvironment and transcriptionally upregulate OTU deubiquitinase 1 (OTUD1) expression, which enhances FGL1 stability via deubiquitination. Disrupting the TAM-OTUD1-FGL1 axis inhibits metastatic tumor progression and synergizes with immune checkpoint blockade (ICB) therapy. Clinically, high plasma FGL1 levels predict poor outcomes and reduced ICB therapy benefits. Benzethonium chloride, an FDA-approved antiseptics, curbs FGL1 secretion, thereby inhibiting liver metastatic tumor growth. Overall, this study uncovers the critical roles and posttranslational regulatory mechanism of FGL1 in promoting metastatic tumor progression, highlighting the TAM-OTUD1-FGL1 axis as a potential target for cancer immunotherapy.

Long noncoding RNA Regulating ImMune Escape regulates mixed lineage leukaemia protein-1-H3K4me3-mediated immune escape in oesophageal squamous cell carcinoma.

BACKGROUND: Predictive biomarkers for oesophageal squamous cell carcinoma (ESCC) immunotherapy are lacking, and immunotherapy resistance remains to be addressed. The role of long noncoding RNA (lncRNA) in ESCC immune escape and immunotherapy resistance remains to be elucidated. METHODS: The tumour-associated macrophage-upregulated lncRNAs and the exosomal lncRNAs highly expressed in ESCC immunotherapy nonresponders were identified by lncRNA sequencing and polymerase chain reaction assays. CRISPR-Cas9 was used to explore the functional roles of the lncRNA. RNA pull-down, MS2-tagged RNA affinity purification (MS2-TRAP) and RNA-binding protein immunoprecipitation (RIP) were performed to identify lncRNA-associated proteins and related mechanisms. In vivo, the humanized PBMC (hu-PBMC) mouse model was established to assess the therapeutic responses of specific lncRNA inhibitors and their combination with programmed cell death protein 1 (PD-1) monoclonal antibody (mAb). Single-cell sequencing, flow cytometry, and multiplex fluorescent immunohistochemistry were used to analyze immune cells infiltrating the tumour microenvironment. RESULTS: We identified a lncRNA that is involved in tumour immune evasion and immunotherapy resistance. High LINC02096 (RIME) expression in plasma exosomes correlates with a reduced response to PD-1 mAb treatment and poor prognosis. Mechanistically, RIME binds to mixed lineage leukaemia protein-1 (MLL1) and prevents ankyrin repeat and SOCS box containing 2 (ASB2)-mediated MLL1 ubiquitination, improving the stability of MLL1. RIME-MLL1 increases H3K4me3 levels in the promoter regions of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1), constitutively increasing the expression of PD-L1/IDO-1 in tumour cells and inhibiting CD8+ T cells infiltration and activation. RIME depletion in huPBMC-NOG mice significantly represses tumour development and improves the effectiveness of PD-1 mAb treatment by activating T-cell-mediated antitumour immunity. CONCLUSIONS: This study reveals that the RIME-MLL1-H3K4me3 axis plays a critical role in tumour immunosuppression. Moreover, RIME appears to be a potential prognostic biomarker for immunotherapy and developing drugs that target RIME may be a new therapeutic strategy that overcomes immunotherapy resistance and benefits patients with ESCC.

[Effect of different suspension moxibustion methods on syndrome characteristics of rats with rheumatoid arthritis of heat bi syndrome based on "moxibustion can be used for heat syndrome"].

OBJECTIVE: To observe the effects of different suspension moxibustion methods on the syndrome characteristics and inflammatory factors of rats with rheumatoid arthritis (RA) of heat bi syndrome and to prove the concept of "moxibustion can be used for heat syndrome". METHODS: Among seventy Wistar rats, 12 rats were randomly selected as a normal group, and the remaining rats were induced by collagen combined with wind, dampness, and heat environmental stimulation to establish the RA model of heat bi syndrome. Forty-eight rats with successful model establishment were further randomly divided into a model group and three moxibustion groups (mild moxibustion group, rotating moxibustion group and sparrow-pecking moxibustion group), with 12 rats in each group. The acupoints "Quchi" (LI 11), "Dazhui" (GV 14) and ashi point were used in all moxibustion groups, with mild moxibustion, rotating moxibustion, and sparrow-pecking moxibustion intervention given respectively, each acupoint was treated with moxibustion for 10 min a day, and 6 days were considered one course of treatment, with a total of three courses. After the intervention, the arthritis index (AI), the Evans blue (EB) extravasated volume in the soft tissue of the right hind paw, and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in the serum were measured by ELISA in each group. The volume of the bilateral hind paw was measured; the infrared thermal imaging was collected to analyze the temperature of the plantar area of the bilateral foot pads, and the reaction time of plantar heat pain was calculated before and after modeling, as well as after the 1st, 2nd and 3rd courses of interrention. The ankle dorsiflexion angle of the right hind foot was also measured before and after modeling, as well as after the intervention. RESULTS: After modeling, compared with the normal group, the rats in the model group had more high-temperature areas in the bilateral hind limbs, abnormal AI score, abnormal bilateral hind paw volume, abnormal temperature of the plantar area of the bilateral foot pads, abnormal foot pain response time, abnormal right hind ankle dorsiflexion angle, abnormal right hind paw soft tissue EB extravasation, and abnormal serum TNF-α and IL-10 levels (P<0.01, P<0.05). After the intervention, compared with the model group, the rats in each moxibustion group had decreased or disappeared high-temperature areas in the bilateral hind limbs, EB extravasated volume in the soft tissue of the right hind paw was reduced (P<0.05), and the right ankle dorsiflexion angle was increased (P<0.05), serum level of TNF-α was reduced, and level of IL-10 increased (P<0.05); the AI scores in the mild moxibustion group and the sparrow-pecking moxibustion group was decreased (P<0.01, P<0.05). After the 1st, 2nd and 3rd courses of intervention, compared with the model group, the bilateral hind paw volume of rats in each moxibustion group was decreased (P<0.05, P<0.01), and plantar heat pain reaction time was increased (P<0.05). After the 2nd course and the 3rd course of intervention, the temperature of the right hind paw pad area was decreased in each moribustion group (P<0.05); after the 3rd courses of intervention, the temperature of the left hind paw pad area was decreased in the mild moxibustion group (P<0.05). CONCLUSION: Suspension moxibustion could adjust the serum levels of TNF-α and IL-10 to improve the syndrome characteristics of RA rats of heat bi syndrome, such as joint redness, swelling, heat, pain and activity restriction. The effect of mild moxibustion is the most prominent. The findings could provide scientific basis for "moxibustion can be used for heat syndrome".

Unveiling the interplay between mutational signatures and tumor microenvironment: a pan-cancer analysis.

Background: While recent studies have separately explored mutational signatures and the tumor microenvironment (TME), there is limited research on the associations of both factors in a pan-cancer context. Materials and methods: We performed a pan-cancer analysis of over 8,000 tumor samples from The Cancer Genome Atlas (TCGA) project. Machine learning methods were employed to systematically explore the relationship between mutational signatures and TME and develop a risk score based on TME-associated mutational signatures to predict patient survival outcomes. We also constructed an interaction model to explore how mutational signatures and TME interact and influence cancer prognosis. Results: Our analysis revealed a varied association between mutational signatures and TME, with the Clock-like signature showing the most widespread influence. Risk scores based on mutational signatures mainly induced by Clock-like and AID/APOBEC activity exhibited strong pan-cancer survival stratification ability. We also propose a novel approach to predict transcriptome decomposed infiltration levels using genome-derived mutational signatures as an alternative approach for exploring TME cell types when transcriptome data are unavailable. Our comprehensive analysis revealed that certain mutational signatures and their interaction with immune cells significantly impact clinical outcomes in particular cancer types. For instance, T cell infiltration levels only served as a prognostic biomarker in melanoma patients with high ultraviolet radiation exposure, breast cancer patients with high homologous recombination deficiency signature, and lung adenocarcinoma patients with high tobacco-associated mutational signature. Conclusion: Our study comprehensively explains the complex interplay between mutational signatures and immune infiltration in cancer. The results highlight the importance of considering both mutational signatures and immune phenotypes in cancer research and their significant implications for developing personalized cancer treatments and more effective immunotherapy.

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis.

Phytase efficiently hydrolyzes phytate to phosphate; thus, it is widely used to increase phosphorus availability in animal feeds and reduce phosphorus pollution through excretion. Phytase is easily inactivated during feed pelleting at high temperature, and sufficient thermostability of phytase is essential for industrial applications. In this study, directed evolution was performed to enhance phytase thermostability. Variants were initially expressed in Escherichia coli BL21 for screening, then in Pichia pastoris for characterization. Over 19,000 clones were generated from an error-prone Polymerase Chain Reaction (epPCR) library; 5 mutants (G10, D7, E3, F8, and F9) were obtained with approximately 9.6%, 10.6%, 11.5%, 11.6%, and 12.2% higher residual activities than the parent after treatment at 99°C for 60 min. Three of these mutants, D7, E3, and F8, exhibited 79.8%, 73.2%, and 92.6% increases in catalytic efficiency (kcat/Km), respectively. In addition, the specific activities of D7, E3, and F8 were 2.33-, 1.98-, and 2.02-fold higher than parental phytase; they were also higher than the activities of all known thermostable phytases. Sequence analysis revealed that all mutants were substituted at residue 75 and was confirmed that the substitution of cysteine at position 75 was the main contribution to the improvement of thermostability of mutants by saturation mutagenesis, indicating that this amino acid is crucial for the stability and catalytic efficiency of phytase. Docking structure analysis revealed that substitution of the C75 residue allowed the mutants to form additional hydrogen bonds in the active pocket, thereby facilitating binding to the substrate. In addition, we confirmed that the intrinsic C77-C108 disulfide bond in E. coli phytase is detrimental to its stability.

An immunogenic and oncogenic feature-based classification for chemotherapy plus PD-1 blockade in advanced esophageal squamous cell carcinoma.

Although chemotherapy plus PD-1 blockade (chemo+anti-PD-1) has become the standard first-line therapy for advanced esophageal squamous cell carcinoma (ESCC), reliable biomarkers for this regimen are lacking. Here we perform whole-exome sequencing on tumor samples from 486 patients of the JUPITER-06 study and develop a copy number alteration-corrected tumor mutational burden that depicts immunogenicity more precisely and predicts chemo+anti-PD-1 efficacy. We identify several other favorable immunogenic features (e.g., HLA-I/II diversity) and risk oncogenic alterations (e.g., PIK3CA and TET2 mutation) associated with chemo+anti-PD-1 efficacy. An esophageal cancer genome-based immuno-oncology classification (EGIC) scheme incorporating these immunogenic features and oncogenic alterations is established. Chemo+anti-PD-1 achieves significant survival improvements in EGIC1 (immunogenic feature-favorable and oncogenic alteration-negative) and EGIC2 (either immunogenic feature-favorable or oncogenic alteration-negative) subgroups, but not the EGIC3 subgroup (immunogenic feature-unfavorable and oncogenic alteration-positive). Thus, EGIC may guide future individualized treatment strategies and inform mechanistic biomarker research for chemo+anti-PD-1 treatment in patients with advanced ESCC.