Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore.

Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis porin A (MspA) nanopore with the N91H substitution, which enables direct identification of all 20 proteinogenic amino acids when combined with a machine-learning algorithm. The validation accuracy reaches 99.1%, with 30.9% signal recovery. The feasibility of ultrasensitive quantification of amino acids was also demonstrated at the nanomolar range. Furthermore, the capability of this system for real-time analyses of two representative post-translational modifications (PTMs), one unnatural amino acid and ten synthetic peptides using exopeptidases, including clinically relevant peptides associated with Alzheimer's disease and cancer neoantigens, was demonstrated. Notably, our strategy successfully distinguishes peptides with only one amino acid difference from the hydrolysate and provides the possibility to infer the peptide sequence.

An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing.

CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.

Effects of CYP3A5*3 genetic polymorphisms on the pharmacokinetics of perampanel in Chinese pediatric patients with epilepsy.

PURPOSE: This study was the first to evaluate the effect of CYP3A5*3 gene polymorphisms on plasma concentration of perampanel (PER) in Chinese pediatric patients with epilepsy. METHODS: We enrolled 98 patients for this investigation. Plasma PER concentrations were measured using liquid chromatography-tandem mass spectrometry. Leftover samples from standard therapeutic drug monitoring were allocated for genotyping analysis. The primary measure of efficacy was the rate of seizure reduction with PER treatment at the final checkup. RESULTS: The plasma concentration showed a linear correlation with the daily dose taken ( r  = 0.17; P  < 0.05). The ineffective group showed a significantly lower plasma concentration of PER (490.5 ±â€…297.1 vs. 633.8 ±â€…305.5 µg/ml; P  = 0.019). For the mean concentration-to-dose (C/D) ratio, the ineffective group showed a significantly lower C/D ratio of PER (3.2 ±â€…1.7 vs. 3.8 ±â€…2.0; P  = 0.040). The CYP3A5*3 CC genotype exhibited the highest average plasma concentration of PER at 562.8 ±â€…293.9 ng/ml, in contrast to the CT and TT genotypes at 421.1 ±â€…165.6 ng/ml and 260.0 ±â€…36.1 ng/ml. The mean plasma PER concentration was significantly higher in the adverse events group (540.8 ±â€…285.6 vs. 433.0 ±â€…227.2 ng/ml; P  = 0.042). CONCLUSION: The CYP3A5*3 gene's genetic polymorphisms influence plasma concentrations of PER in Chinese pediatric patients with epilepsy. Given that both efficacy and potential toxicity are closely tied to plasma PER levels, the CYP3A5*3 genetic genotype should be factored in when prescribing PER to patients with epilepsy.

Metabolic Heterogeneity and Potential Immunotherapeutic Responses Revealed by Single-Cell Transcriptomics of Breast Cancer.

BACKGROUND: Breast cancer (BC) exhibits remarkable heterogeneity. However, the transcriptomic heterogeneity of BC at the single-cell level has not been fully elucidated. METHODS: We acquired BC samples from 14 patients. Single-cell RNA sequencing (scRNA-seq), bioinformatic analyses, along with immunohistochemistry (IHC) and immunofluorescence (IF) assays were carried out. RESULTS: According to the scRNA-seq results, 10 different cell types were identified. We found that Cancer-Associated Fibroblasts (CAFs) exhibited distinct biological functions and may promote resistance to therapy. Metabolic analysis of tumor cells revealed heterogeneity in glycolysis, gluconeogenesis, and fatty acid synthetase reprogramming, which led to chemotherapy resistance. Furthermore, patients with multiple metastases and progression were predicted to benefit from immunotherapy based on a heterogeneity analysis of T cells and tumor cells. CONCLUSIONS: Our findings provide a comprehensive understanding of the heterogeneity of BC, provide comprehensive insight into the correlation between cancer metabolism and chemotherapy resistance, and enable the prediction of immunotherapy responses based on T-cell heterogeneity.

RGS2 attenuates alveolar macrophage damage by inhibiting the Gq/11-Ca2+ pathway during cowshed PM2.5 exposure, and aberrant RGS2 expression is associated with TLR2/4 activation.

Staff and animals in livestock buildings are constantly exposed to fine particulate matter (PM2.5), which affects their respiratory health. However, its exact pathogenic mechanism remains unclear. Regulator of G-protein signaling 2 (RGS2) has been reported to play a regulatory role in pneumonia. The aim of this study was to explore the therapeutic potential of RGS2 in cowshed PM2.5-induced respiratory damage. PM2.5 was collected from a cattle farm, and the alveolar macrophages (NR8383) of the model animal rat were stimulated with different treatment conditions of cowshed PM2.5. The RGS2 overexpression vector was constructed and transfected it into cells. Compared with the control group, cowshed PM2.5 significantly induced a decrease in cell viability and increased the levels of apoptosis and proinflammatory factor expression. Overexpression of RGS2 ameliorated the above-mentioned cellular changes induced by cowshed PM2.5. In addition, PM2.5 has significantly induced intracellular Ca2+ dysregulation. Affinity inhibition of Gq/11 by RGS2 attenuated the cytosolic calcium signaling pathway mediated by PLCß/IP3R. To further investigate the causes and mechanisms of action of differential RGS2 expression, the possible effects of oxidative stress and TLR2/4 activation were investigated. The results have shown that RGS2 expression was not only regulated by oxidative stress-induced nitric oxide during cowshed PM2.5 cells stimulation but the activation of TLR2/4 had also an important inhibitory effect on its protein expression. The present study demonstrates the intracellular Ca2+ regulatory role of RGS2 during cellular injury, which could be a potential target for the prevention and treatment of PM2.5-induced respiratory injury.

Pulsed laser deposition of a Ga2O3 thin film for an optoelectronic synaptic device.

With the rapid development of information era, the traditional von Neumann architecture faces the computing bottleneck, and integration of memory and perception is regarded as a potential solution. Herein, a Ga2O3/Si heterojunction based multi-modulated optoelectronic synaptic device is fabricated and demonstrated. As stimulated by ultraviolet (UV) optical spikes, the heterojunction device reveals typical synaptic functions of excitatory-postsynaptic current (EPSC), paired-pulse facilitation (PPF), spike-timing-dependent plasticity (STDP), and switch between short-term memory (STM) and long-term memory (LTM). In addition, stronger stimulations like higher reading voltage, stronger optical stimulated intensity, and longer pulse duration time can significantly prolong the attenuation of EPSC, which contributes to the improvement of the forgetting process. Our work provides a potential strategy for future neuromorphic computation through a UV light driven stimulation.

Development of growth selection system and pocket engineering of d-amino acid oxidase to enhance selective deamination activity toward d-phosphinothricin.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin ( l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin ( d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

A New Perspective in the Treatment of Ischemic Stroke: Ferroptosis.

Ischemic stroke is a common neurological disease. Currently, there are no Food and Drug Administration-approved drugs that can maximize the improvement in ischemic stroke-induced nerve damage. Hence, treating ischemic stroke remains a clinical challenge. Ferroptosis has been increasingly studied in recent years, and it is closely related to the pathophysiological process of ischemic stroke. Iron overload, reactive oxygen species accumulation, lipid peroxidation, and glutamate accumulation associated with ferroptosis are all present in ischemic stroke. This article focuses on describing the relationship between ferroptosis and ischemic stroke and summarizes the relevant substances that ameliorate ischemic stroke-induced neurological damage by inhibiting ferroptosis. Finally, the problems in the treatment of ischemic stroke targeting ferroptosis are discussed, hoping to provide a new direction for its treatment.

Hydrochar and Its Dissolved Organic Matter Aged in a 30-Month Rice-Wheat Rotation System: Do Primary Aging Factors Alter at Different Stages?

Hydrochar, recognized as a green and sustainable soil amendment, has garnered significant attention. However, information on the aging process in soil and the temporal variability of hydrochar remains limited. This study delves deeper into the interaction between hydrochar and soil, focusing on primary factors influencing hydrochar aging during a 30-month rice-wheat rotation system. The results showed that the initial aging of hydrochar (0-16 months) is accompanied by the development of specific surface area and leaching of hydrochar-derived dissolved organic matter (HDOM), resulting in a smaller particle size and reduced carbon content. The initial aging also features a mineral shield, while the later aging (16 to 30 months) involves surface oxidation. These processes collectively alter the surface charge, hydrophilicity, and composition of aged hydrochar. Furthermore, this study reveals a dynamic interaction between the HDOM and DOM derived from soil, plants, and microbes at different aging stages. Initially, there is a preference for decomposing labile carbon, whereas later stages involve the formation of components with higher aromaticity and molecular weight. These insights are crucial for understanding the soil aging effects on hydrochar and HDOM as well as evaluating the interfacial behavior of hydrochar as a sustainable soil amendment.

Deciphering the Impact of ZnO Nanoparticles and a Sunscreen Product Containing ZnO on Phosphorus Dynamics and Release in Chlorella pyrenoidosa in Aquatic Systems.

Zinc oxide nanoparticles (ZnO NPs) expedite the conversion of organic phosphorus (OP) into PO4-P (Pi), facilitating phosphorus (P) absorption by algae. Our study explored the mechanisms of converting OP (2-aminoethylphosphonic acid (AEP) and ß-glycerol phosphate (ß-GP)) into Pi in Chlorella pyrenoidosa under P deficiency with sunscreen and ZnO NPs. Cell density followed the order of K2HPO4 > ß-GP+ZnO > ß-GP > AEP+ZnO > AEP > P-free. ZnO NPs promoted the conversion of ß-GP, containing C-O-P bonds (0.028-0.041 mg/L), into Pi more efficiently than AEP, which possesses C-P bonds (0.022-0.037 mg/L). Transcriptomics revealed Pi transport/metabolism (phoB (3.99-12.01 fold), phoR (2.20-5.50 fold), ppa (4.49-10.40 fold), and ppk (2.50-5.40 fold)) and phospholipid metabolism (SQD1 (1.85-2.79 fold), SQD2 (2.60-6.53 fold), MGD (2.13-3.21 fold), and DGD (4.08-7.56 fold)) were up-regulated compared to K2HPO4. 31P nuclear magnetic resonance spectroscopy identified intracellular P as polyphosphate, orthophosphate, and pyrophosphate. Synchrotron radiation-based X-ray near-edge structure spectroscopy indicated that K2HPO4 and Zn3(PO4)2 in ß-GP+ZnO were increased by 8.09% and 7.28% compared to AEP+ZnO, suggesting superior P storage in ß-GP+ZnO. Overall, ZnO NPs improved photoinduced electron-hole pair separation and charge separation efficiency and amplified the ·OH and ·O2- levels, promoting OP photoconversion into Pi and algae growth.

Enhancing Phosphorus Release from Sewage Sludge in Anaerobic Digestion via Thermal Hydrolysis Pretreatment: Insights from Phosphorus Speciation and Molecular Biological Pathways.

This study explores the mechanisms enhancing phosphorus (P) release from sludge in anaerobic digestion (AD) with thermal hydrolysis pretreatment (THP) using sequential chemical extraction, X-ray absorption near-edge structure spectroscopy (XANES), 31P NMR, and multiomics. THP-treated sludge notably increased liquid-phase P by 53.8% over 3 days compared to sewage sludge (SS), identifying solid-phase Fe-P as the primary P source. The THP+AD also provided a higher abundance of bacteria that contributed to P release through multiple pathways (MPRPB), whereas SS+AD enriched some microbial species with single P release pathway. Moreover, species co-occurrence network analysis underlined the pivotal role of P-releasing bacteria in THP+AD, with 8 out of 16 keystones being P-releasers. Among the 63 screened genes that were related to P transformations and release, the poly beta-hydroxybutyrate (PHB) synthesis genes associated with polyphosphate bacteria-mediated P release were more abundant in THP+AD than in SS+AD. Furthermore, the upregulation of genes involved in methyl phosphonate metabolism in the THP-treated sludge enhanced the methane production potential of the AD process. These findings suggested that MPRPB were indeed the main contributors to P release, and enrichment in the THP+AD process enhanced their capability for P liberation.

Microbially Driven Iron Cycling Facilitates Organic Carbon Accrual in Decadal Biochar-Amended Soil.

Soil organic carbon (SOC) is pivotal for both agricultural activities and climate change mitigation, and biochar stands as a promising tool for bolstering SOC and curtailing soil carbon dioxide (CO2) emissions. However, the involvement of biochar in SOC dynamics and the underlying interactions among biochar, soil microbes, iron minerals, and fresh organic matter (FOM, such as plant debris) remain largely unknown, especially in agricultural soils after long-term biochar amendment. We therefore introduced FOM to soils with and without a decade-long history of biochar amendment, performed soil microcosm incubations, and evaluated carbon and iron dynamics as well as microbial properties. Biochar amendment resulted in 2-fold SOC accrual over a decade and attenuated FOM-induced CO2 emissions by approximately 11% during a 56-day incubation through diverse pathways. Notably, biochar facilitated microbially driven iron reduction and subsequent Fenton-like reactions, potentially having enhanced microbial extracellular electron transfer and the carbon use efficiency in the long run. Throughout iron cycling processes, physical protection by minerals could contribute to both microbial carbon accumulation and plant debris preservation, alongside direct adsorption and occlusion of SOC by biochar particles. Furthermore, soil slurry experiments, with sterilization and ferrous iron stimulation controls, confirmed the role of microbes in hydroxyl radical generation and biotic carbon sequestration in biochar-amended soils. Overall, our study sheds light on the intricate biotic and abiotic mechanisms governing carbon dynamics in long-term biochar-amended upland soils.

Renal outcomes in IgA nephropathy following inactivated SARS-CoV-2 vaccination.

INTRODUCTION: There are increasing case reports on de novo or relapsing IgA nephropathy (IgAN) following SARS-CoV-2 vaccines, although the follow-up information on renal outcomes in IgAN patients post-SARS-CoV-2 vaccination is limited. In this study, we evaluated the renal outcomes of IgAN patients following inactivated vaccines. METHODS: We investigated the change in eGFR, proteinuria and hematuria in 113 primary IgAN patients post-vaccination. Worsening proteinuria was defined as an increase in proteinuria by more than 0.5 times and proteinuria > 1 g/d. Univariate and multivariable logistic regression analysis were used to evaluate possible predictors of worsening proteinuria. We then compared the renal outcomes of vaccinated patients after 6 months with 101 unvaccinated patients who were followed during the same period. RESULTS: A 2.54% (0.64, 8.61) decrease in renal function was observed in post-vaccination patients. Subgroup analysis revealed a significant decrease in eGFR in patients with 30 ≤ eGFR < 60 (mL/min/1.73 m2) post second SARS-CoV-2 dose (n = 18, p = 0.01). In addition, 10 individuals displayed worsening proteinuria post-vaccination, with the proteinuria subsequently ameliorating significantly after 6-month. Multivariate analysis showed that higher eGFR levels was an independent protective factor for worsening proteinuria. The renal outcome tended towards a decrease in eGFR in vaccinated patients after 6 months follow-up, although the difference was not significant (p = 0.06). CONCLUSION: Kidney function in IgAN patients tended to worsen after SARS-CoV-2 vaccination, particularly those with initial poor kidney function. This pattern of disease flare appears to be clinically mild, and further research is needed to determine whether the impact on kidney function is long-term.

Feasibility of CT attenuation values in distinguishing acute ischemic stroke, old cerebral infarction and leukoaraiosis.

PURPOSE: This study aimed to investigate the feasibility of using computed tomography (CT) attenuation values to differentiate hypodense brain lesions, specifically acute ischemic stroke (AIS) from asymmetric leukoaraiosis (LA) and old cerebral infarction (OCI). MATERIALS AND METHODS: This retrospective study included patients with indeterminate hypodense lesions identified via brain CT scans conducted between June 2019 and June 2021. All lesions were confirmed through head MRI/diffusion-weighted imaging within 48 h after CT. CT attenuation values of hypodense lesions and symmetrical control regions were measured. Additionally, CT attenuation value difference (ΔHU) and ratio (RatioHU) were calculated. One-way analysis of variance (ANOVA) was used to compare age and CT parameters (CT attenuation values, ΔHU and RatioHU) across the groups. Finally, receiver operating characteristic (ROC) analysis was performed to determine the cutoff values for distinguishing hypodense lesions. RESULTS: A total of 167 lesions from 146 patients were examined. The CT attenuation values for AIS(n = 39), LA(n = 53), and OCI(n = 75) were 18.90 ± 6.40 HU, 17.53 ± 4.67 HU, and 11.90 ± 5.92 HU, respectively. The time interval between symptom onset and CT scans for AIS group was 32.21 ± 26.85 h. ANOVA revealed significant differences among the CT parameters of the hypodense lesion groups (all P < 0.001). The AUC of CT values, ΔHU, and RatioHU for distinguishing AIS from OCI were 0.802, 0.896 and 0.878, respectively (all P < 0.001). Meanwhile, the AUC for distinguishing OCI from LA was 0.789, 0.883, and 0.857, respectively (all P < 0.001). Nevertheless, none of the parameters could distinguish AIS from LA. CONCLUSION: CT attenuation parameters can be utilized to differentiate between AIS and OCI or OCI and LA in indeterminate hypodense lesions on CT images. However, distinguishing AIS from LA remains challenging.

Complying with ISO 26262 and ISO/SAE 21434: A Safety and Security Co-Analysis Method for Intelligent Connected Vehicle.

A cyber-physical system (CPS) integrates communication and automation technologies into the operational processes of physical systems. Nowadays, as a complex CPS, an intelligent connected vehicle (ICV) may be exposed to accidental functional failures and malicious attacks. Therefore, ensuring the ICV's safety and security is crucial. Traditional safety/security analysis methods, such as failure mode and effect analysis and attack tree analysis, cannot provide a comprehensive analysis for the interactions between the system components of the ICV. In this work, we merge system-theoretic process analysis (STPA) with the concept phase of ISO 26262 and ISO/SAE 21434. We focus on the interactions between components while analyzing the safety and security of ICVs to reduce redundant efforts and inconsistencies in determining safety and security requirements. To conquer STPA's abstraction in describing causal scenarios, we improved the physical component diagram of STPA-SafeSec by adding interface elements. In addition, we proposed the loss scenario tree to describe specific scenarios that lead to unsafe/unsecure control actions. After hazard/threat analysis, a unified risk assessment process is proposed to ensure consistency in assessment criteria and to streamline the process. A case study is implemented on the autonomous emergency braking system to demonstrate the validation of the proposed method.

Fully digital workflow of occlusal reconstruction treatment in a patient with congenital dentition defects.

OBJECTIVE: Occlusal reconstruction is a critical intervention for patients with dental hard tissue defects, temporomandibular joint (TMJ) disorders, and jaw position abnormalities. Clinical efficiency and outcomes of these procedures have improved with advances in digital technologies. This case report aims to illustrate a comprehensive digital workflow for occlusal reconstruction in a patient with congenital dentition defects, emphasizing the application of digital technologies to enhance treatment outcomes. CLINICAL CONSIDERATIONS: A 28-year-old woman with previously installed porcelain-fused-to-metal bridge restorations presented with a fractured prosthesis and TMJ symptoms. A multidisciplinary approach was adopted involving the use of digital facebow, intraoral scanners, digital smile design, and CAD/CAM technologies. The process included the extraction of defective restorations, temporary restorations to refine jaw position, and final permanent restorations. The digital workflow facilitated precise diagnostics and treatment, culminating in the successful installation of permanent restorations. Regular follow-ups at one- and three-months post-treatment confirmed stable occlusal function and high patient satisfaction. CONCLUSIONS: This case report showcases the potential of multiple digital technologies to streamline complex dental treatments and achieve high-quality results. CLINICAL SIGNIFICANCE: The integration of digital technologies in occlusal reconstruction treatments offers significant benefits in terms of precision, patient comfort, and esthetic outcomes.

Predictors of physical inactivity among pregnant women.

It is recommended that pregnant women be physically active to promote maternal and child health. This study aimed to explore the prevalence of physical inactivity and its modifiable predictors in the three trimesters in Chinese pregnant women. Four hundred forty-four pregnant women completed the Pregnant Physical Activity Questionnaire in the first, second, and third trimesters. The prevalence of physical inactivity reached its highest (66.2%) in the first trimester and lowest (19.4%) in the second trimester. Pregnant women with inadequate physical activity knowledge and low self-efficacy were at higher risk for physical inactivity. Monitoring physical inactivity could be incorporated into antenatal care and start from the first trimester. Prenatal care professionals should take action to increase pregnant women's physical activity self-efficacy and knowledge to enhance their physical activity.

RNA polymerase I subunit D activated by Yin Yang 1 transcription promote cell proliferation and angiogenesis of colorectal cancer cells.

This study aims to explore possible effect of RNA polymerase I subunit D (POLR1D) on proliferation and angiogenesis ability of colorectal cancer (CRC) cells and mechanism herein. The correlation of POLR1D and Yin Yang 1 (YY1) expressions with prognosis of CRC patients in TCGA database was analyzed. Quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot were applied to detect expression levels of POLR1D and YY1 in CRC cell lines and CRC tissues. SW480 and HT- 29 cells were transfected with si-POLR1D or pcDNA3.1-POLR1D to achieve POLR1D suppression or overexpression before cell migration, angiogenesis of human umbilical vein endothelial cells were assessed. Western blot was used to detect expressions of p38 MAPK signal pathway related proteins and interaction of YY1 with POLR1D was confirmed by dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP). TCGA data showed that both POLR1D and YY1 expressions were up-regulated in CRC patients. High expression of POLR1D was associated with poor prognosis of CRC patients. The results showed that POLR1D and YY1 were highly expressed in CRC cell lines. Inhibition or overexpression of POLR1D can respectively suppress or enhance proliferation and angiogenesis of CRC cells. YY1 inhibition can suppress CRC progression and deactivate p38 MAPK signal pathway, which can be counteracted by POLR1D overexpression. JASPAR predicted YY1 can bind with POLR1D promoter, which was confirmed by dual luciferase reporter gene assay and ChIP. YY1 transcription can up-regulate POLR1D expression to activate p38 MAPK signal pathway, thus promoting proliferation and angiogenesis ability of CRC cells.

[Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis].

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

Radical Resection Combined With Intestinal Autotransplantation for Locally Advanced Pancreatic Cancer After Neoadjuvant Therapy: A Report of 36 Consecutive Cases.

OBJECTIVE: To achieve radical resection of locally advanced pancreatic ductal adenocarcinoma (PDAC), and tested the safety and benefits of intestinal autotransplantation in pancreatic surgery. BACKGROUND: PDAC has an extremely dismal prognosis. Radical resection was proved to improve the prognosis of patients with PDAC; however, the locally advanced disease had a very low resection rate currently. We explored and evaluated whether the combination of modern advances in systemic treatment and this macroinvasive surgery was feasible in clinical practice. METHODS: Patients diagnosed as PDAC with superior mesenteric artery involvement and with or without celiac trunk involvement were included. Patients were treated with modified-FOLFIRINOX chemotherapy with or without anti-PD-1 antibodies and were applied to tumor resection combined with intestinal autotransplantation. Data on operative parameters, pathologic results, mortality, morbidity, and survival were analyzed. RESULTS: A total of 36 consecutive cases were applied to this strategy and underwent radical resection combined with intestinal autotransplantation. Among these patients, 24 of them received the Whipple procedure, 11 patients received total pancreatectomy, and the other 1 patient received distal pancreatectomy. The median operation time was 539 minutes. Postoperative pathology showed an R0 resection rate of 94.4%, and tumor invasion of a superior mesenteric artery or superior mesenteric vein was confirmed in 32 patients. The median number of dissected lymph nodes was 43, and 25 patients were positive for lymph node metastasis. The median time of intensive care unit stay was 4 days. Two patients died within 30 days after surgery due to multiorgan failure. The severe postoperative adverse events (equal to or higher than grade 3) were observed in 12 out of 36 patients, and diarrhea, gastroparesis, and abdominal infection were the most frequent adverse events. Postoperative hospital stay was averagely of 34 days. The recurrence-free survival is 13.6 months. The median overall survival of patients after diagnosis and after surgery was 21.4 months and 14.5 months, respectively. CONCLUSIONS: Our attempt suggests the safety of this modality and may be clinically beneficial for highly selected patients with PDAC. However, the experience in multidisciplinary pancreatic cancer care and intestinal transplantation is warranted.