Targeting SOX13 inhibits assembly of respiratory chain supercomplexes to overcome ferroptosis resistance in gastric cancer.

Therapeutic resistance represents a bottleneck to treatment in advanced gastric cancer (GC). Ferroptosis is an iron-dependent form of non-apoptotic cell death and is associated with anti-cancer therapeutic efficacy. Further investigations are required to clarify the underlying mechanisms. Ferroptosis-resistant GC cell lines are constructed. Dysregulated mRNAs between ferroptosis-resistant and parental cell lines are identified. The expression of SOX13/SCAF1 is manipulated in GC cell lines where relevant biological and molecular analyses are performed. Molecular docking and computational screening are performed to screen potential inhibitors of SOX13. We show that SOX13 boosts protein remodeling of electron transport chain (ETC) complexes by directly transactivating SCAF1. This leads to increased supercomplexes (SCs) assembly, mitochondrial respiration, mitochondrial energetics and chemo- and immune-resistance. Zanamivir, reverts the ferroptosis-resistant phenotype via directly targeting SOX13 and promoting TRIM25-mediated ubiquitination and degradation of SOX13. Here we show, SOX13/SCAF1 are important in ferroptosis-resistance, and targeting SOX13 with zanamivir has therapeutic potential.

Chemoproteomics, A Broad Avenue to Target Deconvolution.

As a vital project of forward chemical genetic research, target deconvolution aims to identify the molecular targets of an active hit compound. Chemoproteomics, either with chemical probe-facilitated target enrichment or probe-free, provides a straightforward and effective approach to profile the target landscape and unravel the mechanisms of action. Canonical methods rely on chemical probes to enable target engagement, enrichment, and identification, whereas click chemistry and photoaffinity labeling techniques improve the efficiency, sensitivity, and spatial accuracy of target recognition. In comparison, recently developed probe-free methods detect protein-ligand interactions without the need to modify the ligand molecule. This review provides a comprehensive overview of different approaches and recent advancements for target identification and highlights the significance of chemoproteomics in investigating biological processes and advancing drug discovery processes.

Application and effectiveness of an improved endoscopically guided nasojejunal tube placement technique in critically ill patients: a retrospective cohort study.

Background: The endoscopic nasojejunal (NJ) placement plays a pivotal role in the nutritional support of critically ill patients. However, the conventional endoscopy-guided tube insertion method presents issues of excessive procedural duration. We have enhanced the traditional endoscopy-guided catheter placement method, enabling a faster and more convenient catheter insertion. Methods: We improved the traditional endoscopically guided technique by incorporating an extra silk thread knot at the 25 cm mark on the jejunal segment of the NJ tube to assist endoscopists in accurate tube placement. We conducted the improved NJ tube placement on critically ill patients in need of enteral nutrition (EN). Laboratory data were retrospectively collected before and after the 7-day period of NJ tube placement and EN treatment to evaluate the effectiveness and safety of the improved method. Results: A total of 88 critically ill patients, with an average age of 59.6±15.5 years, and a male ratio of 86.4%, who underwent the improved NJ tube placement method were enrolled into analysis finally, achieving a 100% success rate of NJ tube insertion. The average time for tube insertion was 5.9±2.2 min, with a mean insertion depth of 108.8±12.5 cm. The EN tolerance score was 0.79±0.98. Following 7 days of EN therapy, the patients showed significant improvement in serum albumin levels compared to baseline (36.42 vs. 33.66 g/L, P<0.001). Conclusions: The improved endoscopically guided NJ tube placement technique is a rapid and safe procedure with excellent patient tolerance. It significantly improves the nutritional status of critically ill patients and facilitates the administration of EN, which requires further validation through randomized controlled trials.

Preparation of Microporous Molding Activated Carbon Derived from Bamboo Pyrolysis Gasification Byproducts for Toluene Gas Adsorption.

The effective utilization of charcoal and tar byproducts is a challenge for pyrolysis gasification of bamboo. Herein, the bamboo tar was modified via polymerization and acted as a new adhesive for the preparation of excellent bamboo-charcoal-derived molding activated carbon (MBAC). As compared with pristine tar and other adhesives, the aromatization of tar with phenol increased its molecular weight, oxygenic functional groups, and thermal stability, leading to the decreased blocking impact of charcoal pore and improved bonding and pyrolytic crosslinking effect between charcoal particles. These further contribute to the high mechanical strength, specific surface area, pore volume, and amount of oxygenic functional groups for fabricated MBAC. Owing to the high microporous volume of MBAC, it exhibited 385 mg·g-1 toluene and 75.2% tetrachloride gas adsorption performances. Moreover, the pseudo-first-order, pseudo-second-order, and Bangham models were used to evaluate the kinetic data. The toluene adsorption process conforms to the Bangham kinetic model, suggesting that the diffusion mechanism of toluene adsorption mainly followed intraparticle diffusion.

Revealing the Sequence Characteristics and Molecular Mechanisms of ACE Inhibitory Peptides by Comprehensive Characterization of 160,000 Tetrapeptides.

Chronic diseases, such as hypertension, cause great harm to human health. Conventional drugs have promising therapeutic effects, but also cause significant side effects. Food-sourced angiotensin-converting enzyme (ACE) inhibitory peptides are an excellent therapeutic alternative to pharmaceuticals, as they have fewer side effects. However, there is no systematic and effective screening method for ACE inhibitory peptides, and the lack of understanding of the sequence characteristics and molecular mechanism of these inhibitory peptides poses a major obstacle to the development of ACE inhibitory peptides. Through systematically calculating the binding effects of 160,000 tetrapeptides with ACE by molecular docking, we found that peptides with Tyr, Phe, His, Arg, and especially Trp were the characteristic amino acids of ACE inhibitory peptides. The tetrapeptides of WWNW, WRQF, WFRV, YYWK, WWDW, and WWTY rank in the top 10 peptides exhibiting significantly high ACE inhibiting behaviors, with IC50 values between 19.98 ± 8.19 µM and 36.76 ± 1.32 µM. Salt bridges, π-π stacking, π-cations, and hydrogen bonds contributed to the high binding characteristics of the inhibitors and ACE. Introducing eight Trp into rabbit skeletal muscle protein (no Trp in wide sequence) endowed the protein with a more than 90% ACE inhibition rate, further suggesting that meat with a high content of Trp could have potential utility in hypertension regulation. This study provides a clear direction for the development and screening of ACE inhibitory peptides.

Septin11 promotes hepatocellular carcinoma cell motility by activating RhoA to regulate cytoskeleton and cell adhesion.

Septins as GTPases in the cytoskeleton, are linked to a broad spectrum of cellular functions, including cell migration and the progression of hepatocellular carcinoma (HCC). However, roles of SEPT11, the new member of septin, have been hardly understood in HCC. In the study, the clinical significance and biological function of SEPT11 in HCC was explored. SEPT11 was screened out by combining ATAC-seq with mRNA-seq. Role of SEPT11 in HCC was further investigated by using overexpression, shRNA and CRISPR/Cas9-mediated SEPT11-knockout cells or in vivo models. We found RNA-seq and ATAC-seq highlights LncRNA AY927503 (AY) induced SEPT11 transcription, resulting in Rho GTPase activation and cytoskeleton actin aggregation. The GTP-binding protein SEPT11 is thus considered, as a downstream factor of AY, highly expressed in various tumors, including HCC, and associated with poor prognosis of the patients. In vitro, SEPT11 overexpression promotes the migration and invasion of HCC cells, while SEPT11-knockout inhibits migration and invasion. In vivo, SEPT11-overexpressed HCC cells show high metastasis incidents but don't significantly affect proliferation. Meanwhile, we found SEPT11 targets RhoA, thereby regulating cytoskeleton rearrangement and abnormal cell adhesion through ROCK1/cofilin and FAK/paxillin signaling pathways, promoting invasion and migration of HCC. Further, we found SEPT11 facilitates the binding of GEF-H1 to RhoA, which enhances the activity of RhoA. Overall, our study confirmed function of SEPT11 in promoting metastasis in HCC, and preliminarily explored its related molecular mechanism. SEPT11 acts as an oncogene in HCC, also draws further interest regarding its clinical application as a potential therapeutic target.

Rational Design of a Highly Specific Prolyl Endopeptidase To Activate the Antihypertensive Effect of Peptides.

Enzymatic hydrolysis of food-derived proteins to produce bioactive peptides could activate food functions such as antihypertension. However, the diversity of enzymatic hydrolysis products can reduce bioactive peptides' efficacy. Highly specific proteases can homogenize the hydrolysis products to reduce the production of impotent peptides. In this study, we successfully obtained M. xanthus prolyl endopeptidase mutant Y451M by constraint/free molecular dynamics simulations and binding energy calculations. The specificity of Y451M for proline was increased by 286 % compared to WT, while its activity was almost unchanged. Milk-derived substrates processed with Y451M showed an antihypertensive effect that was 567 % higher than without enzymes. The ability to activate food antihypertension increased 152 % and the use of enzyme by 192 % compared with WT. Specific proteases are thus valuable tools in the processing of complex substrates to obtain bioactive peptides.

Novel management indications for conservative treatment of chylous ascites after gastric cancer surgery.

BACKGROUND: Chylous ascites (CA) presents a challenge as a relatively common postoperative complication in gastric cancer (GC). Primary conservative therapy involved total parenteral nutrition, continuous low-pressure drainage, somatostatin, and a low-fat diet. Drainage tube (DT) clamping has been presented as a potential alternative conservative treatment for GC patients with CA. AIM: To propose novel conservative treatment strategies for CA following GC surgery. METHODS: The data of patients with CA after GC surgery performed at the Fudan University Shanghai Cancer Center between 2006 and 2021 were evaluated retrospectively. RESULTS: 53 patients underwent surgery for GC and exhibited postoperative CA during the study period. Postoperative hospitalization and time of DT removal showed a significant positive association (R 2 = 0.979, P < 0.001). We further observed that delayed DT removal significantly extended the total and postoperative hospitalization, antibiotic usage duration, and hospitalization cost (postoperative hospitalization: 25.8 d vs 15.5 d, P < 0.001; total hospitalization: 33.2 d vs 24.7 d, P < 0.01; antibiotic usage duration: 10.8 d vs 6.2 d, P < 0.01; hospitalization cost: ¥9.2 × 104 vs ¥6.5 × 104, P < 0.01). Multivariate analysis demonstrated that postoperative infection and antibiotic usage were independent factors for delayed DT removal. Furthermore, DT removal times were shorter in seven patients who underwent DT clamping (clamped DT vs normal group, 11.8 d vs 13.6 d, P = 0.047; clamped DT vs delayed group, 13.6 d vs 27.4 d, P < 0.001). In addition, our results indicated that removal of the DT may be possible after three consecutive days of drainage volumes less than 300 mL in GC patients with CA. CONCLUSION: Infection and antibiotic usage were vital independent factors that influenced delayed DT removal in patients with CA. Appropriate standards for DT removal can significantly reduce the duration of hospitalization. Furthermore, DT clamping might be a recommended option for conservative treatment of postoperative CA.

Knockdown of IGF2BP3 inhibits the tumorigenesis of gallbladder cancer and modifies tumor microenvironment.

Gallbladder cancer (GBC) ranks seventh among the gastrointestinal cancers. Messenger RNAs (mRNAs) could regulate the progression of GBC. For the purpose of exploring the targets for GBC treatment, RNA sequencing was used to identify the differential expressed mRNAs between GBC and adjacent tissues. Next, CCK8 assay was used to assess the cell viability, and cell proliferation was investigated by colony formation assay. Flow cytometry was performed to evaluate the cell apoptosis. Protein and mRNA expression were analyzed by western blot and RT-qPCR, respectively. Transwell was performed to evaluate the cell metastasis. GBC-derived exosomes were isolated with ultracentrifugation. To evaluate the function of exosomes in GBC, in vivo model of GBC was constructed. The data revealed IGF2BP3 was identified to be upregulated in GBC, and IGF2BP3 silencing was able to decrease GBC cell proliferation by promoting the apoptosis. The migration and invasion of GBC cells were reduced by IGF2BP3 knockdown. Silencing of IGF2BP3 obviously suppressed the level of p-STAT3 in GBC cells. Meanwhile, GBC cell-derived exosomes notably promoted macrophage M2 polarization via carrying IGF2BP3, and then the polarized macrophages promoted the malignant behavior of GBC cells. Furthermore, exosomes markedly promoted the tumor growth of GBC via promoting macrophage M2 polarization. In summary, knockdown of IGF2BP3 suppressed the malignant behavior of GBC cells. Additionally, knockdown of IGF2BP3 modified tumor microenvironment during the progression of GBC. Thus, these findings might provide a new theoretical basis for exploring a strategies against GBC.

GINS1 promotes the proliferation and migration of glioma cells through USP15-mediated deubiquitination of TOP2A.

GINS1 is a GINS complex subunit that functions along with the MCM2-7 complex and Cdc45 in eukaryotic DNA replication. Despite the significance of the GINS complex in the switch between quiescence and proliferation of glioma cells inside and outside the perinecrotic niche, the biological functions and the underlying mechanism of GINS1 remain unclear. Unlike in normal cells and tissues, GINS1 expression level was significantly upregulated in glioma cells and tissues. High expression of GINS1 predicted an advanced clinical grade and a poor survival. Functional assays revealed that GINS1 aggravated glioma malignant phenotypes in vitro and in vivo. Mechanistically, this study identified that GINS1 physically interacts with TOP2A. GINS1 promotes glioma cell proliferation and migration through USP15-mediated deubiquitination of TOP2A protein. Our results delineate the clinical significance of GINS1 in glioma and the regulatory mechanisms involved in glioma cell proliferation and migration. This work provides potential therapeutic targets for glioma treatment.

Hypoxia inducible lncRNA-CBSLR modulates ferroptosis through m6A-YTHDF2-dependent modulation of CBS in gastric cancer.

Introduction: Tumors are usually refractory to anti-cancer therapeutics under hypoxic conditions. However, the underlying molecular mechanism remains to be elucidated. Objectives: Our study intended to identify hypoxia inducible lncRNAs and their biological function in gastric cancer (GC). Methods: Differentially expressed lncRNAs were determined by microarray analysis between GC cells exposed to hypoxia (1% O2) and normoxia (21% O2) for 24 h. The expression level of CBSLR was manipulated in several GC cell lines to perform molecular and biological analyses both in vitro and in vivo. Results: We identified a hypoxia-induced lncRNA-CBSLR that protected GC cells from ferroptosis, leading to chem-resistance. Mechanically, CBSLR interacted with YTHDF2 to form a CBSLR/YTHDF2/CBS signaling axis that decreased the stability of CBS mRNA by enhancing the binding of YTHDF2 with the m6A-modified coding sequence (CDS) of CBS mRNA. Furthermore, under decreased CBS levels, the methylation of the ACSL4 protein was reduced, leading to protein polyubiquitination and degradation of ACSL4. This, in turn, decreased the pro-ferroptosis phosphatidylethanolamine (PE) (18:0/20:4) and PE (18:0/22:4) content and contributed to ferroptosis resistance. Notably, CBSLR is upregulated, whereas CBS is downregulated in GC tissues compared to matched normal tissues; and GC patients with high CBSLR/low CBS levels have a worse clinical outcome and a poorer response to chemotherapy. Conclusion: Our study reveals a novel mechanism in how HIF1α/CBSLR modulates ferroptosis/chemoresistance in GC, illuminating potential therapeutic targets for refractory hypoxic tumors.

Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry.

The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. These studies collectively further expand our understanding of DYRK2 and provide a valuable tool to pinpoint its biological function.

Activation of MAT2A-ACSL3 pathway protects cells from ferroptosis in gastric cancer.

BACKGROUND: Ferroptosis, a unique form of nonapoptotic-regulated cell death caused by overwhelming lipid peroxidation, represents an emerging tumor suppression mechanism. Growing evidence has demonstrated that cell metabolism plays an important role in the regulation of ferroptosis. Specifically, the association between methionine metabolism and ferroptosis remains undefined. METHODS: We performed in vitro and in vivo experiments to evaluate the influence of methionine metabolism on ferroptosis sensitivity. Pharmacological and genetic blockade of the methionine cycle was utilized and relevant molecular analyses were performed. RESULTS: We identified MAT2A as a driver of ferroptosis resistance. Mechanistically, MAT2A mediates the production of S-adenosylmethionine (SAM), which upregulates ACSL3 by increasing the trimethylation of lysine-4 on histone H3 (H3K4me3) at the promoter area, resulting in ferroptosis resistance. CONCLUSIONS: Collectively, these results established a link between methionine cycle activity and ferroptosis vulnerability in gastric cancer.

CPVL promotes glioma progression via STAT1 pathway inhibition through interactions with the BTK/p300 axis.

CPVL (carboxypeptidase, vitellogenic-like) is a serine carboxypeptidase that was first characterized in human macrophages. However, the function of CPVL remains unclear in a variety of tumors. The quantitative PCR (qPCR), Western blotting, and IHC assays were utilized to measure the CPVL expression. CPVL was significantly upregulated in glioma cells and tissues compared with normal cells and tissues, respectively. Moreover, high CPVL expression was correlated with advanced clinical grade and poor prognosis. Silencing of CPVL promoted glioma cell apoptosis, and it inhibited cell proliferation and tumorigenicity in vitro and in vivo. Ingenuity Pathway Analysis (IPA) demonstrated that CPVL silencing activated the IFN-γ/STAT1 signaling pathway, thereby inducing glioma cell apoptosis. Mechanistically, immunopurification, mass spectrometry, IP, and glutathione S-transferase (GST) pull-down experiments elucidated that CPVL physically interacts with Bruton's tyrosine kinase (BTK) and downregulates the STAT1 phosphorylation through promoting p300-mediated STAT1 acetylation. Our findings reveal the crucial role of CPVL in promoting the progression of glioma through suppressing STAT1 phosphorylation. CPVL might serve as a potential prognostic biomarker and therapeutic target for the treatment of glioma.

Study of the G Protein Nucleolar 2 Value in Liver Hepatocellular Carcinoma Treatment and Prognosis.

LIHC (liver hepatocellular carcinoma) mostly occurs in patients with chronic liver disease. It is primarily induced by a vicious cycle of liver injury, inflammation, and regeneration that usually last for decades. The G protein nucleolar 2 (GNL2), as a protein-encoding gene, is also known as NGP1, Nog2, Nug2, Ngp-1, and HUMAUANTIG. Few reports are shown towards the specific biological function of GNL2. Meanwhile, it is still unclear whether it is related to the pathogenesis of carcinoma up to date. Here, our study attempts to validate the role and function of GNL2 in LIHC via multiple databases and functional assays. After analysis of gene expression profile from The Cancer Genome Atlas (TCGA) database, GNL2 was largely heightened in LIHC, and its overexpression displayed a close relationship with different stages and poor prognosis of carcinoma. After enrichment analysis, the data revealed that the genes coexpressed with GNL2 probably participated in ribosome biosynthesis which was essential for unrestricted growth of carcinoma. Cell functional assays presented that GNL2 knockdown by siRNA in LIHC cells MHCC97-H and SMCC-7721 greatly reduced cell proliferation, migration, and invasion ability. All in all, these findings capitulated that GNL2 could be a promising treatment target and prognosis biomarker for LIHC.

Investigation on the characteristics and mechanisms of ACE inhibitory peptides by a thorough analysis of all 8000 tripeptides via binding free energy calculation.

Food-derived angiotensin I-converting enzyme (ACE) inhibitory peptides represent a potential source of new antihypertensive. However, their characteristics and binding mechanisms were not well understood. In this study, novel energy calculation and experimentation were combined to elucidate the characteristics and mechanisms of ACE inhibitory tripeptides. ACE inhibitory activity of all 8,000 tripeptides was investigated by in silico experiments. IC50 values of the five top-rated tripeptides ranged from 5.86 to 21.84 µM. Five hundred top-ranked tripeptides were chosen for detailed structure-activity analysis, and a significant preference for aromatic amino acids at both C- and N-terminus was found. By binding free energy analysis of nine representative tripeptides via MM/GBSA, electrostatic energy was found to be the leading energy that contributed to the binding of ACE with its high affinity tripeptides. Besides, S355, V380, and V518, three residues positioned around the classical binding pockets of ACE, also played a key role in ACE's binding. Therefore, for tripeptides, their binding pockets in ACE were redefined. In conclusion, the characteristics of ACE inhibitory peptides were more deeply illustrated by the thorough analysis of all tripeptides. The energy analysis allows a better understanding of the binding mechanisms of ACE inhibitory peptides, which could be used to redesign the ACE inhibitors for stronger inhibitory activity.

Drug-guided screening for pancreatic lipase inhibitors in functional foods.

Chronic diseases, such as obesity, cause great harm to human health. Conventional drugs have promising therapeutic effects but also cause significant side effects. Functional foods are an excellent therapeutic alternative to pharmaceuticals, as they have fewer side effects. However, screening for active ingredients in natural foods is difficult. In this study, a novel pancreatic lipase inhibitor screening strategy, guided by the drug molecule orlistat, was combined with experimental verification. Twenty compounds from natural foods were evaluated based on the characteristics of orlistat interaction with pancreatic lipase. The characteristics of 13 molecules were comparable to those of orlistat. The pancreatic lipase inhibition rates of curcumin and sinensetin were 82.42 ± 0.50% and 81.07 ± 2.05%, respectively, and their IC50 values were 0.971 mM and 0.526 mM, respectively; both the inhibition rates as well as IC50 values were similar to those of orlistat. Curcumin and sinensetin prevented weight gain in mice by 69.17% and 52.29%, respectively, compared to orlistat. Curcumin and sinensetin did not cause significant organ damage in vivo, but significantly reduced the contents of triglycerides and cholesterol in blood and lipids in the liver, protecting liver function. Furthermore, 57 328 molecules in the Chinese Natural Product Database library were screened, and 20 potentially active molecules, found to be highly efficient in our study, were selected. Thus, we successfully established an efficient and accurate strategy for screening active ingredients in natural foods under the guidance of a drug molecule, providing valuable insights for functional food development.

Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer.

BACKGROUND: The activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However, the role of methionine metabolism in function of TAMs remains to be explored. METHODS: Monocytes/macrophages were isolated from peripheral blood, tumor tissues or normal tissues from healthy donors or patients with GC. The role of methionine metabolism in the activation of TAMs was evaluated with both in vivo analyses and in vitro experiments. Pharmacological inhibition of the methionine cycle and modulation of key metabolic genes was employed, where molecular and biological analyses were performed. RESULTS: TAMs have increased methionine cycle activity that are mainly attributed to elevated methionine adenosyltransferase II alpha (MAT2A) levels. MAT2A modulates the activation and maintenance of the phenotype of TAMs and mediates the upregulation of RIP1 by increasing the histone H3K4 methylation (H3K4me3) at its promoter regions. CONCLUSIONS: Our data cast light on a novel mechanism by which methionine metabolism regulates the anti-inflammatory functions of monocytes in GC. MAT2A might be a potential therapeutic target for cancer cells as well as TAMs in GC.

Efficacy and mechanism of combination of oxaliplatin with PKM2 knockdown in colorectal cancer.

M2 isomer of pyruvate kinase (PKM2), a key enzyme in aerobic glycolysis, is closely related to cancer development and progression. Suppression of PKM2 exhibits synergistic effects with docetaxel in lung cancer, but the therapeutic potential in colorectal cancer (CRC) is unclear. The aim of the present study was to explore the synergic effects and mechanism of knocking down PKM2 combined with oxaliplatin (a chemosensitizer) treatment in two CRC cell lines (HCT116 and DLD1). The PKM2 gene was initially knocked down using small interfering (si)RNAs (si155 and si156). Subsequently, the effects of PKM2-siRNAs and oxaliplatin, on CRC cells were determined using MTS, cell cycle analysis and apoptosis assays. The mechanism of targeting PKM2 was explored by detecting glucose uptake, lactate secretion fluxes, and the levels of glucose-6-phosphate dehydrogenase (G6PD) mRNA, glutathione (GSH) and reactive oxygen species (ROS). Cell viability in the experimental groups (PKM2-siRNAs, oxaliplatin, PKM2-siRNAs + oxaliplatin) was significantly reduced compared with the control group, and combination treatments (PKM2-siRNAs + oxaliplatin) were more effective than single treatments (PKM2-siRNAs and oxaliplatin only groups). Similar results were observed with the apoptosis assay. The combination groups showed synergistic effects compared with both single treatment groups. Furthermore, glucose uptake and lactate secretion and mRNA levels of G6PD and PKM2 were decreased after PKM2 knockdown in the PKM2-siRNAs and PKM2-siRNAs + oxaliplatin groups. The GSH levels in the PKM2-siRNAs group was significantly lower compared with the negative control group. The ROS levels in the PKM2-siRNAs groups were also significantly increased. The combination of PKM2-siRNAs and oxaliplatin had synergistic effects on CRC cells (HCT116 and DLD1). PKM2 silencing may alter energy metabolism in cancer cells and initiate ROS-induced apoptosis after downregulation of the pentose phosphate pathway by PKM2-siRNAs.

An Energy Optimization Strategy Based on the Perfect Conformation of Prolyl Endopeptidase for Improving Catalytic Efficiency.

Prolyl endopeptidases (PEPs) hydrolyze proteins to yield bioactive peptides and are effective in the treatment of celiac disease. However, the catalytic efficiency of PEPs still has the potential to be improved, which could further strengthen their industrial and therapeutic applications. Herein, a novel rational design strategy based on a "near-attack conformation" of the catalytic state of PEP was adopted. Constrained dynamic simulations were applied, followed by the virtual screening of potentially favorable mutants according to their binding free energy. We redesigned Sphaerobacter thermophiles PEP with high-temperature activity/stability, a wide range of pH stabilities, and high proline specificity. As a result, the kcat value of two PEP mutants (I462W and Q560Y) increased by 208.2 and 150.1%, respectively, and the kcat/KM increased by 32.7 and 6.3%, respectively. These data revealed that the PEP mutants had improved catalytic efficiency and that our strategy can be applied for enzyme engineering.