Advancing MicroRNA Detection: Enhanced Biotin-Streptavidin Dual-Mode Phase Imaging Surface Plasmon Resonance Aptasensor.

MicroRNAs (miRNAs) are novel tumor biomarkers owing to their important physiological functions in cell communication and the progression of multiple diseases. Due to the small molecular weight, short sequence length, and low concentration levels of miRNA, miRNA detection presents substantial challenges, requiring the advancement of more refined and sensitive techniques. There is an urgent demand for the development of a rapid, user-friendly, and sensitive miRNA analysis method. Here, we developed an enhanced biotin-streptavidin dual-mode phase imaging surface plasmon resonance (PI-SPR) aptasensor for sensitive and rapid detection of miRNA. Initially, we evaluated the linear sensing range for miRNA detection across two distinct sensing modalities and investigated the physical factors that influence the sensing signal in the aptamer-miRNA interaction within the PI-SPR aptasensor. Then, an enhanced biotin-streptavidin amplification strategy was introduced in the PI-SPR aptasensor, which effectively reduced the nonspecific adsorption by 20% and improved the limit of detection by 548 times. Furthermore, we have produced three types of tumor marker chips, which utilize the rapid sensing mode (less than 2 min) of PI-SPR aptasensor to achieve simultaneous detection of multiple miRNA markers in the serum from clinical cancer patients. This work not only developed a new approach to detect miRNA in different application scenarios but also provided a new reference for the application of the biotin-streptavidin amplification system in the detection of other small biomolecules.

CRISPR-HOLMES-based NAD+ detection.

Studies have indicated that the intracellular nicotinamide adenine dinucleotide (NAD+) level is associated with the occurrence and development of many diseases. However, traditional nicotinamide adenine dinucleotide (NAD+) detection techniques are time-consuming and may require large and expensive instruments. We recently found that the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a protein can be inactivated by AcrVA5-mediated acetylation and reactivated by CobB, using NAD+ as the co-factor. Therefore, in this study, we created a CRISPR-Cas12a-based one-step HOLMES(NAD+) system for rapid and convenient NAD+ detection with the employment of both acetylated Cas12a and CobB. In HOLMES(NAD+), acetylated Cas12a loses its trans-cleavage activities and can be reactivated by CobB in the presence of NAD+, cutting ssDNA reporters to generate fluorescence signals. HOLMES(NAD+) shows both sensitivity and specificity in NAD+ detection and can be used for quantitative determination of intracellular NAD+ concentrations. Therefore, HOLMES(NAD+) not only provides a convenient and rapid approach for target NAD+ quantitation but also expands the application scenarios of HOLMES to non-nucleic acid detection.

The identification of a novel compound heterozygous mutation in hereditary human coagulation factor VII deficiency following a bamboo leaf green snake bite.

Hereditary factor VII (FVII) deficiency is an uncommon autosomal recessive disorder associated with mutations in the F7 gene, and laboratory investigations usually reveal isolated prolongation in prothrombin time (PT)/international normalized ratio (INR). Venom-induced consumptive coagulopathy (VICC) is distinguished by the activation of the coagulation pathway, which is triggered by procoagulant toxins in snake venom. Diagnosing snakebites in patients with hereditary FVII deficiency presents a challenge because prolonged time PT/INR is considered the most valuable diagnostic method for VICC. Therefore, it is possible that certain patients may not promptly receive an accurate diagnosis of hereditary FVII deficiency. We present a pedigree featuring hereditary FVII deficiency, which was diagnosed through Sanger sequencing, following a bamboo leaf green snake bite.

Induction sintilimab and chemotherapy followed by concurrent chemoradiotherapy for locally advanced esophageal cancer: a proof-of-concept, single-arm, multicenter, phase 2 trial.

Background: Concurrent chemoradiotherapy is the standard nonoperative treatment for locally advanced esophageal squamous cell carcinoma. However, local recurrence is still the main failure pattern, accounting for more than half of all treatment failures, indicating that the sensitivity of radiotherapy still needs to be improved. This trial aimed at demonstrating whether PD-1 inhibitors followed by chemoradiotherapy could promote esophageal tumor vascular normalization, alleviate hypoxia, and thus enhance radiosensitivity and improve local control. Methods: We did a multicenter, single-arm, phase 2 trial in China. Patients with locally advanced esophageal cancer were enrolled in this study. In induction phase, patients received two cycles of sintilimab, paclitaxel and carboplatin once per 21 days. In concurrent phase, patients were treated with five cycles of carboplatin and paclitaxel once per week concurrent with radiotherapy of 50.4Gy delivered in 28 fractions. The primary endpoint was 2-year local control rate. Hypoxia and vessel normalization was assessed before and after induction phase using immunofluorescence and perfusion CT. This trial is registered with ClinicalTrials.gov (NCT03985046). Findings: Seventy-five patients with esophageal cancer were enrolled in this study between October 2019 and April 2021. The median follow-up of surviving patients was 33.6 months (IQR 29.3-35.7). The 2-year local control rate was 81.7% (95% confidence interval, 72.7%-90.7%), which was much higher than that in concurrent chemoradiation only (71.3%) in previous studies. Vascular normalization and hypoxia alleviation were observed in both biopsy specimens and perfusion CT. Interpretation: The addition of induction immunotherapy to standard concurrent chemoradiotherapy could improve radiosensitivity for locally advanced esophageal cancer as non-surgical treatment. New treatment combination led to higher local control rate through promoting vascular normalization and alleviating hypoxia. Our findings suggest that induction immunotherapy followed by concurrent chemoradiotherapy could be a potential option in future treatment. Funding: National Natural Science Foundation of China and Shanghai Rising-Star Program.

meHOLMES: A CRISPR-cas12a-based method for rapid detection of DNA methylation in a sequence-independent manner.

Aberrant DNA methylation is closely associated with various diseases, particularly cancer, and its precise detection plays an essential role in disease diagnosis and monitoring. In this study, we present a novel DNA methylation detection method (namely meHOLMES), which integrates both the TET2/APOBEC-mediated cytosine deamination step and the CRISPR-Cas12a-based signal readout step. TET2/APOBEC efficiently converts unmethylated cytosine to uracil, which is subsequently changed to thymine after PCR amplification. Utilizing a rationally designed crRNA, Cas12a specifically identifies unconverted methylated cytosines and generates detectable signals using either fluorescent reporters or lateral flow test strips. meHOLMES quantitatively detects methylated CpG sites with or without Protospacer Adjacent Motif (PAM) sequences in both artificial and real biological samples. In addition, meHOLMES can complete the whole detection process within 6 h, which is much faster than traditional bisulfite-based sample pre-treatment method. Above all, meHOLMES provides a simpler, faster, more accurate, and cost-effective approach for quantitation of DNA methylation levels in a sequence-independent manner.

Research progress on GlnR-mediated regulation in Actinomycetes.

This review constitutes a summary of current knowledge on GlnR, a global regulator, that assumes a critical function in the regulation of nitrogen metabolism of Actinomycetes. In cross-regulation with other regulators, GlnR was also shown to play a role in the regulation of carbon and phosphate metabolisms as well as of secondary metabolism. A description of the structure of the GlnR protein and of its binding sites in various genes promoters regions is also provided. This review thus provides a global understanding of the critical function played by GlnR in the regulation of primary and secondary metabolism in Actinomycetes.

Impact of hematological and radiation parameters on the clinical prognosis of esophageal cancer patients treated with definitive chemoradiotherapy.

This study aimed to conduct a survival analysis of thoracic esophageal squamous cell carcinoma (ESCC) patients treated with radical chemoradiotherapy and identify prognostic variables from among the hematological and radiation parameters. Cases of patients with ESCC receiving definitive chemoradiotherapy at Jiangsu Cancer Hospital between January 2018 and September 2020 were screened. A Cox proportional hazards model was used to assess the effect of hematological and radiation parameters on the overall survival (OS). The neutrophil-to-lymphocyte ratio (NLR) was calculated by dividing the absolute neutrophil count (ANC) by the absolute lymphocyte count (ALC) in the week prior to radical chemoradiotherapy. Variables associated with radiation were gathered based on dose-volume histograms (DVH). X-tile software was used to determine the optimal cutoff values for pretreatment NLR and posttreatment ALC nadir. Associations between lymphopenia and dose-volume parameters were analyzed using multivariate logistic regression. The study included 104 ESCC patients. The median follow-up of surviving patients was 45.0 months (interquartile range: 40.2-52.2), with 1- and 3-year OS rates of 88.0% and 62.7%, respectively. Multivariate Cox regression analysis demonstrated a significant survival benefit in patients with low baseline NLR (≤ 2.2), high ALC nadir (> 0.24*109/L), and desirable radiation parameters for the heart and thoracic vertebrae. Increased dose-volume parameters of the heart, lungs, and thoracic vertebrae were correlated with a high probability of radiation-induced lymphopenia (RIL) risk (P < 0.05). Baseline NLR and RIL are significantly related to survival outcomes in ESCC patients. Optimization of radiation parameters of cardiopulmonary and thoracic vertebrae can be effective in the prevention of RIL.

Cytoplasm Hydrogelation-Mediated Cardiomyocyte Sponge Alleviated Coxsackievirus B3 Infection.

Viral myocarditis (VMC), commonly caused by coxsackievirus B3 (CVB3) infection, lacks specific treatments and leads to serious heart conditions. Current treatments, such as IFNα and ribavirin, show limited effectiveness. Herein, rather than inhibiting virus replication, this study introduces a novel cardiomyocyte sponge, intracellular gelated cardiomyocytes (GCs), to trap and neutralize CVB3 via a receptor-ligand interaction, such as CAR and CD55. By maintaining cellular morphology, GCs serve as sponges for CVB3, inhibiting infection. In vitro results revealed that GCs could inhibit CVB3 infection on HeLa cells. In vivo, GCs exhibited a strong immune escape ability and effectively inhibited CVB3-induced viral myocarditis with a high safety profile. The most significant implication of this study is to develop a universal antivirus infection strategy via intracellular gelation of the host cell, which can be employed not only for treating defined pathogenic viruses but also for a rapid response to infection outbreaks caused by mutable and unknown viruses.

Pseudoprogression in the era of immunotherapy-based strategies for recurrent head and neck squamous cell carcinoma achieving complete response: A case report.

RATIONALE: In the last few years, treatment of head and neck squamous cell carcinoma (HNSCC) has been enhanced by the emergence of immunotherapy. A biological phenomenon unique to immunotherapy is pseudoprogression, an increase in tumor burden or the appearance of a new lesion subsequently followed by tumor regression. PATIENT CONCERNS: A 78-year-old man complaining of a lump (6*4 cm) gradually swelling on the right side of his neck with recurrent buccal mucosa squamous cell carcinoma presented to our institution. Two months prior, he received resection of the buccal lesion but refused suggested adjuvant chemoradiotherapy after the operation. DIAGNOSES: Recurrent buccal mucosa squamous cell carcinoma. INTERVENTIONS: Induction immunotherapy was initiated, followed by a new node appearing on the surface of the neck mass. We considered the presence of pseudoprogression and continued with immunotherapy. The patient received immunotherapy combined with chemotherapy and intensity-modulated radiation therapy (IMRT) consecutively. OUTCOMES: The patient experienced an excellent recovery with the disappearance of pain and the lump, along with return of a healthy appetite, weight gain and positive outlook. Complete response (CR) was also noted by magnetic resonance imaging (MRI) scan, with the upper right neck mass significantly retreated to unclear display. The patient is still alive with stable, asymptomatic disease at the time of this writing. LESSONS: These results provide confidence in the safety and efficacy of radical chemo-radio-immunotherapy for the treatment of recurrent, unresectable or metastatic HNSCC.

Antibacterial Activity and Mechanism of Canagliflozin against Methicillin-Resistant Staphylococcus aureus.

Diabetic foot infection (DFI) is a common complication in diabetes patients, with foot infections being the leading cause of amputations. Staphylococcus aureus is frequently found in diabetic foot infections, of which methicillin-resistant Staphylococcus aureus (MRSA) has become a major clinical and epidemiological challenge. Since MRSA strains are resistant to most ß-lactam antibiotics, and also partially resistant to other antibiotics, treatment is difficult and costly. The emergence of drug-resistant bacteria often arises from overuse or misuse of antibiotics. Clinically, canagliflozin is commonly used for the treatment of type 2 diabetes. On this basis, we investigated the antibacterial activity and mechanism of canagliflozin against MRSA, with the aim to discover novel functions of canagliflozin and provide new insights for the treatment of MRSA. Using the microbroth dilution method to determine the half maximal inhibitory concentration of drugs, we found that canagliflozin not only can inhibit the growth of methicillin-sensitive Staphylococcus aureus (MSSA) but also exhibits antibacterial activity against MRSA. The IC50 values, at approximately 56.01 µM and 57.60 µM, were almost the same. At 12 h, canagliflozin showed a significant antibacterial effect against MRSA at and above 30 µM. In addition, its combined use with penicillin achieved better antibacterial effects, which were increased by about three times. Additive antibacterial activity (FICI = 0.69) was found between penicillin and canagliflozin, which was better than that of doxycycline and canagliflozin (FICI = 0.95). Canagliflozin also affected bacterial metabolic markers, such as glucose, ATP, and lactic acid. The results of crystal violet staining indicate that canagliflozin disrupted the formation of bacterial biofilm. Our electron microscopy results showed that canagliflozin distorted the bacterial cell wall. The results of RT-PCR suggest that canagliflozin down-regulated the expressions of biofilm-related gene (clfA, cna, agrC, mgrA, hld) and methicillin-resistance gene (mecA), which was related to MRSA. Molecular docking also indicated that canagliflozin affected some interesting targets of MRSA, such as the sarA, crtM and fnbA proteins. In conclusion, canagliflozin exhibits antibacterial activity against MRSA by affecting bacterial metabolism, inhibiting its biofilm formation, distorting the bacterial cell wall, and altering the gene expression of biofilm formation and its virulence. Our study reveals the antibacterial activity of canagliflozin against MRSA, providing a new reference for treating diabetic foot infections.

Peptide aptamer-based time-resolved fluoroimmunoassay for CHIKV diagnosis.

BACKGROUND: Chikungunya virus (CHIKV) and Dengue virus (DENV) have similar clinical symptoms, which often induce misdiagnoses. Therefore, an antigen detection diagnostic system that can clearly identify these two viruses is desirable. METHODS: In this study, we developed a novel peptide with high affinity and specificity to CHIKV, and further constructed peptide aptamer-based TRFIA assay to efficiently detect CHIKV. Peptide aptamer B2 (ITPQSSTTEAEL) and B3 (DTQGSNWI) were obtained through computer-aided design and selected as CHIKV-specific peptide aptamers based on their high binding affinity, strong hydrogen bonding, and RMSD of molecular docking. Then, a sandwich-Time-Resolved Fluoroimmunoassay (TRFIA) was successfully constructed for the detection of the interaction between peptide aptamers and viruses. RESULTS: When using B2 as the detection element, highly specific detection of CHIKV E2 was achieved with detection limits of 8.5 ng/ml in PBS solution. Variation coefficient between inter-assay showed the disturbances received from the detection of clinical fluid specimens (including serum and urine), were also within acceptable limits. The detection limits for 10-fold dilution serum and urine were 57.8 ng/mL and 147.3 ng/mL, respectively. The fluorescent signal intensity exhibited a good linear correlation with E2 protein concentration in the range of 0-1000 ng/mL, indicating the potential for quantitative detection of E2 protein. CONCLUSIONS: These results demonstrate that the construction of peptide aptamers with high affinity and specificity provides an excellent method for rapid diagnostic element screening, and the developed peptide aptamer B2 contributed to better detection of CHIKV viral particles compared to traditional antibodies.

Intracellular hydrogelation of macrophage conjugated probiotics for hitchhiking delivery and combined treatment of colitis.

Immune cell membrane coated nanomedicine was developed to neutralize cytokines via receptor-ligand interaction, which showed potential for the treatment of inflammatory bowel disease (IBD). However, cell membrane isolation and re-assembly process involved protein loss and spatial disorder, which reduced the sequestration efficiency towards cytokines. In addition, oral administration of probiotics was accepted for IBD treatment via gut microbiota modulation, but most probiotics showed weak adhesion to intestine mucosa and were quickly expelled from gastrointestinal tract. Herein, an intracellular hydrogelation technology was proposed to construct gelated peritoneal macrophage (GPM) with intact membrane structure, resulting from the avoidance of membrane isolation and re-assembly process. GPM efficiently neutralized multiple cytokines in vitro and in vivo to ameliorate inflammatory Caco-2 â€‹cells and colitis rats by regulating oxidative stress, inflammation level and intestinal barrier repair. Moreover, the probiotics (Nissle1917, EcN) were easily attached on GPM surface through specific recognition, to construct GPM-EcN conjugate for GPM hitchhiking delivery to colitis tissue. Conjugation process of GPM and EcN showed no damage on bacterial physiological function. Due to the chemical attachment on inflammatory cells, GPM carried the attached EcN hand-in-hand to accumulate in the colitis tissue of IBD rat, and enhanced intestine retention time of EcN in comparison to free EcN, which improved bacterial diversity, and shifted the microbiota community and acid metabolites to an anti-inflammatory phenotype. This study transferred the hydrogel synthesis from in vitro to intracellular cytoplasm, and came to a new insight of conjugating strategy of GPM and probiotics for hitchhiking delivery and combined anti-IBD treatment.

CRISPR-Cas12-Based Diagnostic Applications in Infectious and Zoonotic Diseases.

Rapid detection of infectious and zoonotic diseases is very important for pathogen identification and infection control. Molecular diagnostic assays are well-known for high accuracy and sensitivity; however, conventional methods such as real-time PCR may require professional instruments and operations, preventing their wide applications in scenarios including animal quarantine. The recently developed CRISPR diagnostic (CRISPR-Dx) methods, employing the trans-cleavage activities of either Cas12 (e.g., HOLMES) or Cas13 (e.g., SHERLOCK), have shown great potential in rapid and convenient nucleic acid detection. Guided by specially designed CRISPR RNA (crRNA), Cas12 binds target DNA sequences and trans-cleaves ssDNA reporters, generating detectable signals, while Cas13 recognizes target ssRNA and trans-cleaves ssRNA reporters. To achieve high detection sensitivity, both HOLMES and SHERLOCK systems can be combined with pre-amplification procedures including both PCR and isothermal amplifications. Here, we present the employment of the HOLMESv2 method for convenient detection of the infectious and zoonotic diseases. Specifically, target nucleic acid is first amplified by LAMP or RT-LAMP, and the products are then detected by the thermophilic Cas12b. In addition, Cas12b reaction can be combined with LAMP amplification to achieve one-pot reaction systems. In this chapter, we provide a step-by-step description of the HOLMESv2-mediated rapid and sensitive detection of Japanese encephalitis virus (JEV), an RNA pathogen as an example.

Removing Negative Impacts from Inevitable Nonreproducible and Nonspecific Antibody-Probe Interactions in Viral Serology.

Serological assays are indispensable tools in public health. Presently deployed serological assays, however, largely overlook research progress made in the last two decades that jeopardizes the conceptual foundation of these assays, i.e., antibody (Ab) specificity. Challenges to traditional understanding of Ab specificity include Ab polyspecificity and most recently nonreproducible Ab-probe interactions (NRIs). Here, using SARS-CoV-2 and four common livestock viruses as a test bed, we developed a new serological platform that integrates recent understanding about Ab specificity. We first demonstrate that the response rate (RR) from a large-sized serum pool (∼100) is not affected by NRIs or by nonspecific Ab-probe interactions (NSIs), so RR can be incorporated into the diagnostic probe selection process. We subsequently used multiple probes (configured as a "protein peptide hybrid microarray", PPHM) to generate a digital microarray index (DMI) and finally demonstrated that DMI-based analysis yields an extremely robust probabilistic trend that enables accurate diagnosis of viral infection that overcomes multiple negative impacts exerted by NSI/NRI. Thus, our study with SARS-CoV-2 confirms that the PPHM-RR-DMI platform enables very rapid development of serological assays that outperform traditional assays (for both sensitivity and specificity) and supports that the platform is extendable to other viruses.

Human liver cancer organoids: Biological applications, current challenges, and prospects in hepatoma therapy.

Liver cancer and disease are among the most socially challenging global health concerns. Although organ transplantation, surgical resection and anticancer drugs are the main methods for the treatment of liver cancer, there are still no proven cures owing to the lack of donor livers and tumor heterogeneity. Recently, advances in tumor organoid technology have attracted considerable attention as they can simulate the spatial constructs and pathophysiological characteristics of tumorigenesis and metastasis in a more realistic manner. Organoids may further contribute to the development of tailored therapies. Combining organoids with other emerging techniques, such as CRISPR-HOT, organ-on-a-chip, and 3D bioprinting, may further develop organoids and address their bottlenecks to create more practical models that generalize different tissue or organ interactions in tumor progression. In this review, we summarize the various methods in which liver organoids may be generated and describe their biological and clinical applications, present challenges, and prospects for their integration with emerging technologies. These rapidly developing liver organoids may become the focus of in vitro clinical model development and therapeutic research for liver diseases in the near future.

An Integrated ddPCR Lab-on-a-Disc Device for Rapid Screening of Infectious Diseases.

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient-dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection.

Neoadjuvant sintilimab and chemotherapy in patients with resectable esophageal squamous cell carcinoma: A prospective, single-arm, phase 2 trial.

Background: Immunotherapy (Programmed cell death 1 blockade) has entered the ranks of advanced esophageal cancer first-line treatment; however, little is known about the efficacy of PD-1 inhibitor as neoadjuvant therapy in resectable esophageal squamous cell carcinoma (ESCC). We aim to evaluate the activity and safety of the neoadjuvant sintilimab combined with chemotherapy in the treatment of resectable thoracic ESCC. Methods: The enrolled patients with resectable (clinical stage II to IVA) ESCC received neoadjuvant sintilimab injection (200 mg/time, day 1), paclitaxel liposomes (135 mg/m2, day 1), and carboplatin (area under curve of 5 mg/mL/min, day 1) every 21 days for 2 cycles, and esophagectomy was performed within 3-6 weeks after the 2 cycles of treatment. The primary endpoint of the study was the pathological complete response (PCR) rate. Results: From July 2019 to March 2021, a total of 47 patients were enrolled, of which 33 patients (70.2%) had clinical stage III disease. All patients completed the full two-cycle treatment and forty-five patients received radical surgery, including 44 (97.8%) R0 resections. Ten (22.2%) of 45 patients had a PCR, and the major pathological response (MPR) rate was 44.4% (20/45). The grade 3-4 treatment-related adverse events (TRAEs) were mainly neutropenia (6 of 47,12.8%) and leucopenia (8 of 47,17.0%). One (2.1%) patient occurred postoperative immune-associated encephalitis. No delays in surgery were observed. Conclusions: sintilimab combined with paclitaxel liposome and carboplatin, as demonstrated in this phase II trial to exhibit a relatively high PCR rate and acceptable safety, warrants additional investigation in resectable ESCC. Trial Registration: http://www.chictr.org.cn/, ChiCTR1900026593.

DDX56 transcriptionally activates MIST1 to facilitate tumorigenesis of HCC through PTEN-AKT signaling.

Rationale: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver that is the leading cause of cancer-related mortality worldwide. However, genetic alterations and mechanisms underlying HCC development remain unclear. Methods: Tissue specimens were used to evaluate the expression of DEAD-Box 56 (DDX56) to determine its prognostic value. Colony formation, CCK8, and EdU-labelling assays were performed to assess the effects of DDX56 on HCC proliferation. The in vivo role of DDX56 was evaluated using mouse orthotopic liver xenograft and subcutaneous xenograft tumor models. Dual-luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were performed to examine the effect of DDX56 on the MIST1 promoter. Results: DDX56 expression in HCC tissues was elevated and this increase was strongly correlated with poor prognoses for HCC patients. Functionally, DDX56 promoted HCC cell proliferation both in vitro and in vivo, while mechanistically interacting with MECOM to promote HCC proliferation by mono-methylating H3K9 (H3K9me1) on the MIST1 promoter, leading to enhanced MIST1 transcription and subsequent regulation of the PTEN/AKT signaling pathway, which promotes HCC proliferation. More importantly, the PTEN agonist, Oroxin B (OB), blocked the DDX56-mediated PTEN-AKT signaling pathway, suggesting that treating HCC patients with OB may be beneficial as a therapeutic intervention. Furthermore, we observed that ZEB1 bound to DDX56 and transcriptionally activated DDX56, leading to HCC tumorigenesis. Conclusions: Our results indicated that the ZEB1-DDX56-MIST1 axis played a vital role in sustaining the malignant progression of HCC and identified DDX56 as a potential therapeutic target in HCC tumorigenesis.

Postoperative Concurrent Chemoradiotherapy for Locally Advanced Thoracic Esophageal Squamous Cell Carcinoma: A Phase II Clinical Trial.

Background: This study aims to investigate the efficacy and safety of postoperative intensity-modulated radiotherapy (IMRT) covering partial regional lymph node areas combined with chemotherapy for locally advanced thoracic esophageal squamous cell carcinoma patients. Methods: This was a single-center, single-arm phase II clinical trial that began in 2014. Patients who underwent radical transthoracic resection within 3 months and were histologically confirmed esophageal squamous cell carcinoma (pT3-4 or N+, M0 determined according to AJCC Guidelines, Edition 7) were recruited. Postoperative radiotherapy was performed with a total dose of 50.4Gy in 28 fractions using IMRT. Clinical target volumes (CTVs) included tumor bed, anastomosis, bilateral supraclavicular region, and superior mediastinal lymph nodes. Synchronous chemotherapy for 2 cycles (paclitaxel 150mg/m2, day1; Cisplatin 25mg/m2, day1-3; every 4 weeks), followed by 2 cycles of consolidation chemotherapy with the same regimen. The primary endpoint was the 2-year local control rate, and the secondary endpoints were overall survival (OS) and adverse events (AEs). Results: A total of 75 eligible patients were included from 2014 to 2017. The 2-year LRFS rate, as the primary endpoint, was 73.3%. The 1-year and 3-year OS rates were 88.0% and 68.0%, respectively. Local recurrence occurred in 13/75 (17.4%) patients, of which 2.7% (2/75) were extra-target lymph nodes. Grade 4 adverse events reported in this study included 10 cases (13.3%) of neutropenia, 1 case (1.3%) of anemia, and 2 cases (2.7%) of thrombocytopenia, without toxic-related deaths. Almost all (96%) patients completed the entire postoperative radiotherapy course, and 62 (82.7%) patients completed at least 2 cycles of chemotherapy. Conclusion: Postoperative IMRT (clinical target volume including tumor bed, anastomosis, bilateral supraclavicular region, and superior mediastinal lymph nodes) combined with synchronous chemotherapy in patients with locally advanced thoracic esophageal squamous cell carcinoma was well tolerated, with a high local control rate and a low probability of recurrence outside the irradiation field. Clinical Trial Registration: https://clinicaltrials.gov/ChiCTR1900022689.

Paclitaxel and cisplatin combined with concurrent involved-field irradiation in definitive chemoradiotherapy for locally advanced esophageal squamous cell carcinoma: a phase II clinical trial.

PURPOSE: This trial aims to explore the efficacy and safety of involved-field irradiation (IFI) combined with paclitaxel plus cisplatin as concurrent chemoradiotherapy for locally advanced esophageal squamous cell carcinoma (ESCC), under the premise of intensity-modulated radiotherapy (IMRT). METHODS: Enrolled patients with locally advanced ESCC were treated with definitive concurrent chemoradiotherapy. IFI was administered adopting IMRT and the total dose was 61.2 Gy delivered in 34 fractions. On the first day of radiotherapy, the patients were treated with paclitaxel and cisplatin one cycle per month for 2 cycles followed by the same regimen in consolidation chemotherapy for two cycles. The primary endpoint of the study was the 2-year locoregional recurrence-free survival (LRFS) rate, and secondary endpoints included overall survival (OS), progression-free survival (PFS), and safety. RESULTS: Between January 2018 and September 2020, 108 patients participated in the trial. 78.7% (85/108) of patients completed all 4 cycles of chemotherapy. The median follow-up of the surviving patients was 33.9 months (interquartile range, 29.2-41.1). The 2-year LRFS rate, as the primary endpoint, was 64.2%. In addition, the median PFS was 39.2 months, and 1-year and 3-year OS rates of 88.0% and 63.3%, respectively. Among the patients, out-of-field regional failure was seen in only 7 (6.5%) patients. Neutropenia grade 3 and 4 occurred in 21.3% and 37.0% of the patients, respectively. CONCLUSIONS: IFI using IMRT combined with paclitaxel and cisplatin concurrent chemotherapy for locally advanced ESCC yields encouraging local control and overall survival, but high hematological toxicity. Trial registration Clinical Trials ChiCTR1800017039.