Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.

Personalized prediction of postoperative complication and survival among Colorectal Liver Metastases Patients Receiving Simultaneous Resection using machine learning approaches: A multi-center study.

BACKGROUND: To predict clinical important outcomes for colorectal liver metastases (CRLM) patients receiving colorectal resection with simultaneous liver resection by integrating demographic, clinical, laboratory, and genetic data. METHODS: Random forest (RF) models were developed to predict postoperative complications and major complications (binary outcomes), as well as progression-free survival (PFS) and overall survival (OS) (time-to-event outcomes) of the CRLM patients based on data from two hospitals. The models were validated on an external dataset from an independent hospital. The clinical utility of the models was assessed via decision curve analyses (DCA). RESULTS: There were 1067 patients included in survival prediction analyses and 1070 patients included in postoperative complication prediction analyses. The RF models provided an assessment of the model contributions of features for outcomes and suggested KRAS, BRAF, and MMR status were salient for the PFS or OS predictions. RF model of PFS showed that the Brier scores at 1-, 3-, and 5-year PFS were 0.213, 0.202 and 0.188; and the AUCs of 1-, 3- and 5-year PFS were 0.702, 0.720 and 0.743. RF model of OS revealed that Brier scores of 1-,3-, and 5-year OS were 0.040, 0.183 and 0.211; and the AUCs of 1-, 3- and 5-year OS were 0.737, 0.706 and 0.719. RF model for postoperative complication resulted in an AUC of 0.716 and a Brier score of 0.196. DCA curves clearly demonstrated that the RF models for these outcomes exhibited a superior net benefit across a wide range of threshold probabilities, signifying their favorable clinical utility. The RF models consistently exhibited robust performance in both internal cross-validation and external validation. The individualized risk profile predicted by the models closely aligned with the actual survival outcomes observed for the patients. A web-based tool (https://kanli.shinyapps.io/CRLMRF/) was provided to demonstrate the practical use of the prediction models for new patients in the clinical setting. CONCLUSION: The predictive models and a web-based tool for personalized prediction demonstrated a moderate predictive performance and favorable clinical utilities on several key clinical outcomes of CRLM patients receiving simultaneous resection, which could facilitate the clinical decision-making and inform future interventions for CRLM patients.

Surgical treatment improves overall survival of hepatocellular carcinoma with extrahepatic metastases after conversion therapy: a multicenter retrospective study.

Systemic therapy is typically the primary treatment choice for hepatocellular carcinoma (HCC) patients with extrahepatic metastases. Some patients may achieve partial response (PR) or complete response (CR) with systemic treatment, leading to the possibility of their primary tumor becoming resectable. This study aimed to investigate whether these patients could achieve longer survival through surgical resection of their primary tumor. We retrospectively collected data from 150 HCC patients with extrahepatic metastases treated at 15 different centers from January 1st, 2015, to November 30th, 2022. We evaluated their overall survival (OS) and progress-free survival (PFS) and analyzed risk factors impacting both OS and PFS were analyzed. Patients who received surgical treatment had longer OS compared to those who did not (median OS 16.5 months vs. 11.3 months). However, there was no significant difference in progression-free survival between the two groups. Portal vein invasion (P = 0.025) was identified as a risk factor for poor prognosis in patients, while effective first-line treatment (P = 0.039) and surgical treatment (P = 0.005) were protective factors. No factors showed statistical significance in the analysis of PFS. Effective first-line treatment (P = 0.027) and surgical treatment (P = 0.006) were both independent protective factors for prolonging patient prognosis, while portal vein invasion was an independent risk factor (P = 0.044). HCC patients with extrahepatic metastases who achieve PR/CR with conversion therapy may experience longer OS through surgical treatment. This study is the first to analyze the clinical outcomes of patients receiving surgical treatment for HCC with extrahepatic metastases.

MiR-380 inhibits the proliferation and invasion of cholangiocarcinoma cells by silencing LIS1.

BACKGROUND: The objective of this study was to determine the role and regulatory mechanism of miR-380 in cholangiocarcinoma. METHODS: The TargetScan database and a dual-luciferase reporter assay system were used to determine if LIS1 was a target gene of miR-380. The Cell Counting Kit 8 assay, flow cytometry, and Transwell assay were used to detect the effects of miR-380 and LIS1 on the proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. Western blotting was used to determine the effect of miR-380 on MMP-2/p-AKT. Immunohistochemistry detected the regulatory effect of miR-380 on the expression of MMP-2/p-AKT/LIS1. RESULTS: Expression of miR-380 in cholangiocarcinoma was decreased but expression of LIS1 was increased. LIS1 was confirmed to be a target gene of miR-380. Transfection with miR-380 mimics inhibited the proliferation, S-phase arrest, and invasion of HCCC-9810/HuCCT1/QBC939 cells, and LIS1 reversed these inhibitory effects. miR-380 inhibitor promoted proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. si-LIS1 salvaged the promotive effect of miR-380 inhibitor. Overexpression of miR-380 inhibited expression of MMP-2/p-AKT/LIS1, but miR-380 inhibitor promoted their expression. CONCLUSION: An imbalance of miR-380 expression is closely related to cholangiocarcinoma, and overexpression of miR-380 inhibits the expression of MMP-2/p-AKT by directly targeting LIS1.

FOLFOXIRI plus cetuximab as conversion therapy for unresectable RAS/BRAF wild-type left-sided colorectal cancer with liver-limited metastases: a prospective dual-center pilot study.

Purpose: To explore the efficacy and safety of FOLFOXIRI plus cetuximab regimen as conversion therapy for patients with unresectable RAS/BRAF wild-type colorectal liver-limited metastases (CLM). Patients and methods: This was a dual-center, phase II trial with the rate of no evidence of disease (NED) achieved as the primary endpoint. All enrolled patients with initially unresectable left-sided RAS/BRAF wild-type colorectal liver-limited metastases received a modified FOLFOXIRI plus cetuximab regimen as conversion therapy. Results: Between October 2019 and October 2021, fifteen patients were enrolled. Nine patients (60%) achieved NED. The overall response rate (ORR) was 92.9%, and the disease control rate (DCR) was 100%. The median relapse-free survival (RFS) was 9 (95% CI: 0-20.7) months. The median progression-free survival (PFS) was 13.0 months (95% CI: 5.7-20.5), and the median overall survival (OS) was not reached. The most frequently occurring grade 3-4 adverse events were neutropenia (20%), peripheral neurotoxicity (13.3%), diarrhea (6.7%), and rash acneiform (6.7%). Conclusion: The FOLFOXIRI plus cetuximab regimen displayed tolerable toxicity and promising anti-tumor activity in terms of the rate of NED achieved and response rate in patients with initially unresectable left-sided RAS/BRAF wild-type CLM. This regimen merits further investigation.

Tumor burden score and carcinoembryonic antigen predict outcomes in patients with intrahepatic cholangiocarcinoma following liver resection: a multi­institutional analysis.

BACKGROUND: The prognostic significance of tumor burden score (TBS) in relation to carcinoembryonic antigen (CEA) has not been investigated among patients undergoing hepatectomy for intrahepatic cholangiocarcinoma (ICC). This study aimed to develop and validate a simplified model, a combination of TBS and CEA (CTC grade), for predicting the long-term outcomes of postoperative ICC patients. METHODS: Patients who underwent curative - intent resection of ICC between 2011 and 2019 were identified from a large multi - institutional database. The impact of TBS, CEA, and the CTC grade on overall survival (OS) and recurrence - free survival (RFS) was evaluated in both the derivation and validation cohorts. The receiver operating characteristic curve was utilized for assessing the predictive accuracy of the model. Subgroup analyses were performed across 8th TNM stage system stratified by CTC grade to assess the discriminatory capacity within the same TNM stage. RESULTS: A total of 812 patients were included in the derivation cohort and 266 patients in the validation cohort. Survival varied based on CEA (low: 36.7% vs. high: 9.0%) and TBS (low: 40.3% vs. high: 17.6%) in relation to 5 - year survival (both p < 0.001). As expected, patients with low CTC grade (i.e., low TBS/low CEA) were associated with the best OS as well as RFS, while high CTC grade (i.e., high TBS/high CEA) correlated to the worst outcomes. The model exhibited well performance in both the derivation cohort (area under the curve of 0.694) and the validation cohort (0.664). The predictive efficacy of the CTC grade system remains consistently stable across TNM stages I and III/IV. CONCLUSION: The CTC grade, a composite parameter derived from the combination of TBS and CEA levels, served as an easy - to - use tool and performed well in stratifying patients with ICC relative to OS and RFS.

HBV integrations reshaping genomic structures promote hepatocellular carcinoma.

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.

Drugs targeting TGF-ß/Notch interaction attenuate hypertrophic scar formation by optic atrophy 1-mediated mitochondrial fusion.

Hypertrophic scar (HS) formation is a cutaneous fibroproliferative disease that occurs after skin injuries and results in severe functional and esthetic disability. To date, few drugs have shown satisfactory outcomes for the treatment of HS formation. Transforming growth factor-beta (TGF-ß)/Notch interaction via small mothers against decapentaplegic 3 (Smad3) could facilitate HS formation; therefore, targeting TGF-ß/ Notch interaction via Smad3 is a potential therapeutic strategy to attenuate HS formation. In addition, optic atrophy 1 (OPA1)-mediated mitochondrial fusion contributes to fibroblast proliferation, and TGF-ß/Smad3 axis and the Notch1 pathway facilitate OPA1-mediated mitochondrial fusion. Thus, the aim of this study was to investigate whether drugs targeting TGF-ß/Notch interaction via Smad3 suppressed fibroblast proliferation to attenuate HS formation through OPA1-mediated mitochondrial fusion. We found that the TGF-ß pathway, Notch pathway, and TGF-ß/Notch interaction via Smad3 were inhibited by pirfenidone, the gamma- secretase inhibitor DAPT, and SIS3 in human keloid fibroblasts (HKF) and an HS rat model, respectively. Protein interaction was detected by co-immunoprecipitation, and mitochondrial morphology was determined by electron microscopy. Our results indicated that pirfenidone, DAPT, and SIS3 suppressed the proliferation of HKFs and attenuated HS formation in the HS rat model by inhibiting TGF-ß/Notch interaction via Smad3. Moreover, pirfenidone, DAPT, and SIS3 hindered OPA1-mediated mitochondrial fusion through inhibiting TGF-ß/Notch interaction, thereby suppressing the proliferation of HS fibroblasts and HS formation. In summary, these findings investigating the effects of drugs targeting TGF-ß/Notch interaction on HS formation might lead to novel drugs for the treatment of HS formation.

Anatomic versus non-anatomic resection for early-stage intrahepatic cholangiocarcinoma: a propensity score matching and stabilized inverse probability of treatment weighting analysis.

BACKGROUND: Radical resection is still the most cost-effectiveness curative strategy for intrahepatic cholangiocarcinoma (ICC), but it remains controversial on the survival benefit of anatomic resection (AR). In this study, we sought to compare the oncologic outcomes between AR versus non-AR (NAR) as the primary treatment for early-stage ICC patients. METHODS: Data of ICC patients who underwent hepatectomy and staged at AJCC I were retrospectively collected from 12 hepatobiliary centers in China between Dec 2012 and Dec 2015. Propensity score matching (PSM) and stabilized inverse probability of treatment weighting (IPTW) analysis were performed to minimize the effect of potential confounders, and the perioperative and long-term outcomes between AR and NAR groups were compared. RESULTS: Two hundred seventy-eight ICC patients staged at AJCC I were eligible for this study, including 126 patients receiving AR and 152 patients receiving NAR. Compared to the NAR group, the AR group experienced more intraoperative blood loss before and after PSM or stabilized IPTW (all P > 0.05); AR group also experienced more intraoperative transfusion after stabilized IPTW (P > 0.05). In terms of disease-free survival (DFS) and overall survival (OS), no significant differences were observed between the two groups before and after PSM or stabilized IPTW (all P > 0.05). Multivariable Cox regression analyses found that AR was not an independent prognostic factor for either DFS or OS (all P > 0.05). Further analysis also showed that the survival benefit of AR was not found in any subgroup stratified by Child-Pugh grade (A or B), cirrhosis (presence or absence), tumor diameter (≤ 5 cm or > 5 cm) and pathological type (mass-forming or non-mass-forming) with all P > 0.05. CONCLUSION: Surgical approach does not influence the prognosis of patients with stage I primary ICC, and NAR might be acceptable and oncological safety.

The comparison of three different molecular imaging methods in localization and grading of insulinoma.

Aims: This cross-sectional study compared the value of molecular imaging (Exendin-4 positron emission tomography/computed tomography [PET/CT], 68Ga-DOTATATE PET/CT, 18F- fluorodeoxyglucose [FDG] PET/CT) in insulinoma localization by stratified tumor size and grading, and explored the correlation of the related the maximum standardized uptake value (SUVmax) with insulinoma grading, Ki-67, maximum tumor diameter, and glucose metabolism. Methods: In 28 insulinoma patients, the sensitivity of three types of PET/CT for localizing insulinoma was calculated according to tumor size and grade. We compared the SUVmax for different insulinoma grades and analyzed the correlation of SUVmax with Ki-67, maximum tumor diameter, and glucose metabolism indicators. Results: The study included 12 grade (G) 1 and 16 G2 cases, with maximum tumor diameters ranging from 9 to 40 mm. Without differentiation by size and grade, the sensitivity of Exendin-4 PET/CT to localize insulinoma was 100%, which significantly exceeded that of 68Ga-DOTATATE PET/CT and 18F-FDG PET/CT (75% and 57%, respectively). In tumors with a maximum diameter ≤ 20 mm and ≤ 15 mm, the sensitivity of Exendin-4 (both 100%) significantly exceeded that of 68Ga-DOTATATE PET/CT (74% and 64%, respectively) and 18F-FDG PET/CT (54% and 50%, respectively). In G1 tumors, the sensitivity of Exendin-4 PET/CT was significantly higher than that of 18F-FDG PET/CT, but not that of 68Ga-DOTATATE PET/CT, while in G2 tumors, the sensitivity of Exendin-4 PET/CT was significantly higher than that of both other types. However, all three PET/CT types missed a metastatic lymph node in one patient. The 18F-FDG PET/CT SUVmax was significantly lower than that of the other PET/CT types and that of 68Ga-DOTATATE PET/CT was significantly lower in G2 than in G1. 68Ga-DOTATATE PET/CT SUVmax correlated negatively with Ki-67. A receiver operating characteristic (ROC) curve suggested that 68Ga-DOTATATE PET/CT SUVmax > 19.9 could predict G1 tumors. Conclusion: Exendin-4 PET/CT was superior to 68Ga-DOTATATE PET/CT and 18F-FDG PET/CT for insulinoma localization, particularly small and G2 tumors, but its diagnostic value in small metastatic lymph nodes requires further exploration. 68Ga-DOTATATE PET/CT SUVmax could be used as an adjunct to pathology, and a value > 19.9 could predict G1 tumors. No PET/CT SUVmax could predict tumor maximum diameter and glucose metabolism.

An elevated preoperative cholesterol-to-lymphocyte ratio predicts unfavourable outcomes in colorectal cancer liver metastasis patients receiving simultaneous resections: a retrospective study.

BACKGROUND: To explore the clinical prognostic utility of the preoperative cholesterol-to-lymphocyte ratio (CLR) in outcomes for colorectal cancer liver metastasis (CRLM) patients receiving simultaneous resection of the primary lesion and liver metastases. METHODS: A total of 444 CRLM patients receiving simultaneous resections were enrolled. The optimal cut-off value for CLR was determined using the highest Youden's index. Patients were divided into the CLR < 3.06 group and the CLR≥3.06 group. Propensity score matching analysis (PSM) and the inverse probability of treatment weighting (IPTW) method were conducted to eliminate bias between the two groups. The outcomes included short-term outcomes and long-term outcomes. Kaplan-Meier curves and log-rank tests were used to analyse progression-free survival (PFS) and overall survival (OS). RESULTS: In the short-term outcome analysis, after 1:1 PSM, 137 patients were distributed to the CLR < 3.06 group and CLR≥3.06 group. No significant difference was noted between the two groups (P > 0.1). Compared with patients with CLR < 3.06, patients with CLR≥3.06 had comparable operation times (320.0 [272.5-421.0] vs. 360.0 [292.5-434.5], P = 0.088), blood loss (200.0 [100.0-400.0] vs. 200.0 [150.0-450.0], P = 0.831), postoperative complication rates (50.4% vs. 46.7%, P = 0.546) and postoperative ICU rates (5.8% vs. 11.7%, P = 0.087). In the long-term outcome analysis, Kaplan-Meier analysis showed that compared with patients with CLR < 3.06, patients with CLR≥3.06 had worse PFS (P = 0.005, median: 10.2 months vs. 13.0 months) and OS (P = 0.002, median: 41.0 months vs. 70.9 months). IPTW-adjusted Kaplan-Meier analysis showed that the CLR≥3.06 group had worse PFS (P = 0.027) and OS (P = 0.010) than the CLR < 3.06 group. In the IPTW-adjusted Cox proportional hazards regression analysis, CLR≥3.06 was an independent factor for PFS (HR = 1.376, 95% CI 1.097-1.726, P = 0.006) and OS (HR = 1.723, 95% CI 1.218-2.439, P = 0.002). IPTW-adjusted Cox proportional hazards regression analysis including postoperative complications, operation time, intraoperative blood loss, intraoperative blood transfusion and postoperative chemotherapy revealed that CLR≥3.06 was an independent factor for PFS (HR = 1.617, 95% CI 1.252-2.090, P < 0.001) and OS (HR = 1.823, 95% CI 1.258-2.643, P = 0.002). CONCLUSIONS: The preoperative CLR level predicts unfavourable outcomes in CRLM patients receiving simultaneous resection of the primary lesion and liver metastases and should be taken into consideration when developing treatment and monitoring strategies.

Nomogram for tumour response based on prospective cohorts of hepatocellular carcinoma patients receiving immunotherapy combined with targeted therapy: development and validation.

Background: Although immunotherapy combined with targeted therapy can be effective for hepatocellular carcinoma (HCC), not all HCC patients respond to this treatment. Models for predicting tumour response in HCC patients receiving immunotherapy combined with targeted therapy are lacking. Methods: A total of 221 HCC patients from two independent prospective cohorts were retrospectively reviewed. The patients were randomly divided into training and validation cohorts at a ratio of 7:3. Standard clinical data were collected from each patient, including age, sex, hepatitis B infection status, laboratory tests, and immune target-related adverse events (itrAEs). Tumour responses were evaluated using the Response Evaluation Criteria in Solid Tumours (RECIST) v1.1 guidelines. ItrAEs were assessed based on the Common Terminology Criteria for Adverse Events version 4.0. The nomogram for tumour response prediction was constructed based on the results of the multivariate logistic regression analysis, areas under the receiver operating characteristic curves (AUROCs) were used to determine the sensitivity and specificity of the model, and calibration plots and Hosmer-Lemeshow chi-square tests were performed to assess the calibration of the model. Results: In the multivariate logistic regression analysis, a solitary tumour (P=0.006), neutropenia (P=0.003) and hypertension (P=0.042) independently predicted objective response (OR). A nomogram for OR was established with AUROCs of 0.734, 0.675, 0.730, and 0.707 in the training, validation, first-line and second-line treatment sets, respectively. Tumour sizes less than 5 cm (P=0.005), a solitary tumour (P=0.037), prognostic nutritional indices greater than or equal to 54.3 (P=0.037), neutropenia (P=0.004) and fatigue (P=0.041) independently predicted disease control (DC). A nomogram for DC was established with AUROCs of 0.804, 0.667, and 0.768 in the training, first-line and second-line treatment sets, respectively. All the Hosmer-Lemeshow tests and calibration curves showed acceptable calibration. Conclusions: The current provides clinicians with new insights into selecting patients for immunotherapy combined with targeted therapy and contributes to the development of immunotherapy for HCC. It is necessary to expand the scale of our research and perform prospective studies to verify our findings.

Prognostic significance of the preoperative alkaline phosphatase­to­albumin ratio in patients with hepatocellular carcinoma after hepatic resection.

This study aimed to investigate the prognostic value of the preoperative alkaline phosphatase-to-albumin ratio (APAR) in patients with hepatocellular carcinoma (HCC) who underwent radical hepatectomy. The clinicopathological data from 330 patients was retrospectively analyzed. Receiver operating characteristic curves of APAR for diagnostic tumor recurrence were plotted with a cut-off value of 1.74. A high preoperative APAR value was significantly associated with hepatitis B surface antigen level, tumor diameter, and tumor-node-metastasis stage. The disease-free survival (DFS) and overall survival (OS) of patients with a high preoperative APAR were shorter than those with a low APAR. The independent risk factors for DFS were an APAR ≥1.74, and macrovascular invasion or tumor thrombus. The independent risk factors for OS were an APAR ≥1.74, existing clinical symptoms, α-fetoprotein level ≥20 ng/ml, macrovascular invasion or tumor thrombus, and family history of cancer. In conclusion, a preoperative APAR (≥1.74) is an independent risk factor influencing the poor prognosis of patients with HCC after curative hepatectomy, and patients with such a result should be closely monitored.

etiBsu1209: A comprehensive multiscale metabolic model for Bacillus subtilis.

Genome-scale metabolic models (GEMs) have been widely used to guide the computational design of microbial cell factories, and to date, seven GEMs have been reported for Bacillus subtilis, a model gram-positive microorganism widely used in bioproduction of functional nutraceuticals and food ingredients. However, none of them are widely used because they often lead to erroneous predictions due to their low predictive power and lack of information on regulatory mechanisms. In this work, we constructed a new version of GEM for B. subtilis (iBsu1209), which contains 1209 genes, 1595 metabolites, and 1948 reactions. We applied machine learning to fill gaps, which formed a relatively complete metabolic network able to predict with high accuracy (89.3%) the growth of 1209 mutants under 12 different culture conditions. In addition, we developed a visualization and code-free software, Model Tool, for multiconstraints model reconstruction and analysis. We used this software to construct etiBsu1209, a multiscale model that integrates enzymatic constraints, thermodynamic constraints, and transcriptional regulatory networks. Furthermore, we used etiBsu1209 to guide a metabolic engineering strategy (knocking out fabI and yfkN genes) for the overproduction of nutraceutical menaquinone-7, and the titer increased to 153.94 mg/L, 2.2-times that of the parental strain. To the best of our knowledge, etiBsu1209 is the first comprehensive multiscale model for B. subtilis and can serve as a solid basis for rational computational design of B. subtilis cell factories for bioproduction.

Artificial intelligence technologies in bioprocess: Opportunities and challenges.

Bioprocess control and optimization are crucial for tapping the metabolic potential of microorganisms, and which have made great progress in the past decades. Combination of the current control and optimization technologies with the latest computer-based strategies will be a worth expecting way to improve bioprocess further. Recently, artificial intelligence (AI) emerged as a data-driven technique independent of the complex interactions used in mathematical models and has been gradually applied in bioprocess. In this review, firstly, AI-guided modeling approaches of bioprocess are discussed, which are widely applied to optimize critical process parameters (CPPs). Then, AI-assisted rapid detection and monitoring technologies employed in bioprocess are summarized. Next, control strategies according to the above two technologies in bioprocess are analyzed. Lastly, current research gaps and future perspectives on AI-guided optimization and control technologies are discussed. This review provides theoretical guidance for developing AI-guided bioprocess optimization and control technologies.

Dynamic evaluation of postoperative survival in intrahepatic cholangiocarcinoma patients who did not undergo lymphadenectomy: a multicenter study.

BACKGROUND: The prognosis of Intrahepatic cholangiocarcinoma (ICC) patients who did not undergo lymphadenectomy is difficult to assess. This study aims to have a dynamic evaluation on the postoperative survival of ICC patients by calculating conditional survival. METHODS: Relevant data were from patients treated in 12 large-scale hospitals from December 2011 to December 2017. The influence of relevant clinical baseline data on the prognosis of ICC patients was analyzed by Cox regression. Conditional survival (CS) is a method that may predict the prognostic probability dynamically. For a patient with x years of survival, the 1-year CS (CS1) may be calculated as CS1= OS(x + 1)/OS(x). RESULT: A total of 361 patients who met the criteria were included in the study. Conditional survival (CS) means that the patients' prognosis varies with survival time, meanwhile, relevant factors affecting the prognosis have a time-varying effect. The probability of survival assessed by CS1 increased year by year and the 1,2,3-year survival improved from 68.4% to 87.8%, while the postoperative actuarial OS decreased from 69.4% at 1 years to 36.9% at 3 years. CONCLUSIONS: In terms of CS, the estimated survival for ICC varies with the increase of survival time after excision. Patients who live longer were likely to live longer. At the same time, with the passage of time, the role of the original adverse factors of the tumor would gradually decrease. Conditional survival allows a more accurate assessment of ICC patients who did not undergo lymphadenectomy.

Machine learning for metabolic pathway optimization: A review.

Optimizing the metabolic pathways of microbial cell factories is essential for establishing viable biotechnological production processes. However, due to the limited understanding of the complex setup of cellular machinery, building efficient microbial cell factories remains tedious and time-consuming. Machine learning (ML), a powerful tool capable of identifying patterns within large datasets, has been used to analyze biological datasets generated using various high-throughput technologies to build data-driven models for complex bioprocesses. In addition, ML can also be integrated with Design-Build-Test-Learn to accelerate development. This review focuses on recent ML applications in genome-scale metabolic model construction, multistep pathway optimization, rate-limiting enzyme engineering, and gene regulatory element designing. In addition, we have discussed some limitations of these methods as well as potential solutions.

Research progress of spontaneous ruptured hepatocellular carcinoma: Systematic review and meta-analysis.

Background: Spontaneously ruptured hepatocellular carcinoma (rHCC) with hemorrhage is characterized by rapid onset and progression. The aim of this systematic review was to explore the current studies on rHCC with hemorrhage and determine the optimum treatment strategy. Method: The PubMed, Web of Science, Embase, and the Cochrane Library databases were searched for studies reporting survival outcomes with comparison between emergency resection (ER) and transarterial embolization following staged hepatectomy (SH) were included by inclusion and exclusion criteria, the perioperative and survival data were statistically summarized using Review Manager 5.3 software. Result: A total of 8 retrospective studies were included, with a total sample size of 556, including 285 (51.3%) in the ER group and 271 (48.7%) in the SH group. The perioperative blood loss and blood transfusion volume in the SH group were less than those in the ER group, and there were no significant differences in the operative time, incidence of complications, mortality and recurrence rate of tumors between the two groups. The 1-, 2-, 3-year overall survival and 1-, 2-, 3-, 5-year disease-free survival of the ER group were not significantly different from those of the SH group, and the 5-year overall survival rate of ER group was lower than that of the SH group (hazard ratios=1.52; 95% confidence intervals: 1.14-2.03, P=0.005). Conclusion: There was no significant difference in the short-term efficacy of ER or SH in the treatment of ruptured HCC, and SH was superior to ER in the long-term survival.

Optimal surgical sequence for colorectal cancer liver metastases patients receiving colorectal cancer resection with simultaneous liver metastasis resection: A multicentre retrospective propensity score matching study.

BACKGROUND: There is little evidence regarding the optimal surgical sequence for colorectal cancer liver metastasis (CRLM) patients undergoing colorectal resection with simultaneous liver metastasis resection. METHODS: CRLM patients from five centers were retrospectively evaluated. The short-term outcomes included intraoperative and postoperative outcomes. Postoperative complications were measured according to the Clavien-Dindo classification. Grade I to II complications were defined as minor postoperative complications. The long-term outcomes were progression-free survival (PFS) and overall survival (OS). Propensity score matching (PSM) and inverse probability of treatment weighting (IPTW) were performed to overcome the selection bias between colorectal resection first and liver resection first. RESULTS: A total of 1255 CRLM patients were included. In the multivariable logistic regression analysis, a body mass index (BMI) < 24 kg/m2, primary site in the left hemicolon, non-bilobar distribution of liver metastases and no preoperative chemotherapy were significantly associated with the likelihood of colorectal resection first. After 1:1 PSM, there was no significant difference between the colorectal resection first group and the liver resection first group. Compared with patients with colorectal resection first, patients with liver resection first had a comparable postoperative infection rate (15.0% vs. 16.0%, P = 0.735), a longer operation time (305.0 [231.3-416.0] vs. 300.0 [225.0-374.0], P = 0.033), more intraoperative blood loss (200.0 [150.0-400.0] vs. 100.0 [100.0-300.0], P < 0.001), a higher postoperative minor complication rate (28.7% vs. 20.7%, P = 0.023) and a higher postoperative ICU rate (14.7% vs. 8.7%, P = 0.022). IPTW-adjusted Kaplan-Meier analysis showed that patients who underwent colorectal resection first had a similar PFS (P = 0.702, median: 20.6 months vs. 16.6 months) and unfavourable OS (P = 0.014, median: 48.5 months vs. 67.0 months) compared with patients who underwent liver resection first. In the IPTW-adjusted Cox proportional hazards regression analysis, colorectal resection first was an unfavourable risk factor for OS (hazard ratio [HR] = 1.301, 95% CI 1.048-1.616, P = 0.017) and was not an independent predictor for PFS (HR = 0.986, 95% CI 0.831-1.170, P = 0.874). IPTW-adjusted Cox proportional hazards regression analysis, including postoperative complications, operation time, intraoperative blood loss and postoperative chemotherapy, produced consistent results. CONCLUSION: Although violating the "sterility principle", patients who underwent colorectal resection first did not have an increased postoperative infection rate and had some better short-term outcomes and comparable PFS than those who underwent liver resection first.