Identification and validation of COL6A1 as a novel target for tumor electric field therapy in glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive primary brain malignancy. Novel therapeutic modalities like tumor electric field therapy (TEFT) have shown promise, but underlying mechanisms remain unclear. The extracellular matrix (ECM) is implicated in GBM progression, warranting investigation into TEFT-ECM interplay. METHODS: T98G cells were treated with TEFT (200 kHz, 2.2 V/m) for 72 h. Collagen type VI alpha 1 (COL6A1) was identified as hub gene via comprehensive bioinformatic analysis based on RNA sequencing (RNA-seq) and public glioma datasets. TEFT intervention models were established using T98G and Ln229 cell lines. Pre-TEFT and post-TEFT GBM tissues were collected for further validation. Focal adhesion pathway activity was assessed by western blot. Functional partners of COL6A1 were identified and validated by co-localization and survival analysis. RESULTS: TEFT altered ECM-related gene expression in T98G cells, including the hub gene COL6A1. COL6A1 was upregulated in GBM and associated with poor prognosis. Muti-database GBM single-cell analysis revealed high-COL6A1 expression predominantly in malignant cell subpopulations. Differential expression and functional enrichment analyses suggested COL6A1 might be involved in ECM organization and focal adhesion. Western blot (WB), immunofluorescence (IF), and co-immunoprecipitation (Co-IP) experiments revealed that TEFT significantly inhibited expression of COL6A1, hindering its interaction with ITGA5, consequently suppressing the FAK/Paxillin/AKT pathway activity. These results suggested that TEFT might exert its antitumor effects by downregulating COL6A1 and thereby inhibiting the activity of the focal adhesion pathway. CONCLUSION: TEFT could remodel the ECM of GBM cells by downregulating COL6A1 expression and inhibiting focal adhesion pathway. COL6A1 could interact with ITGA5 and activate the focal adhesion pathway, suggesting that it might be a potential therapeutic target mediating the antitumor effects of TEFT.

The CONSTANS-LIKE gene PeCOL13 regulates flowering through intron-retained alternative splicing in Phyllostachys edulis.

Woody bamboo exhibits a unique flowering characteristic with a lengthy flowering cycle, often followed by death. In many plant species, alternative splicing (AS) is a common phenomenon involved in controlling flowering. In this study, a PeCOL13 gene in moso bamboo (Phyllostachys edulis) was characterized. It produced two isoforms: PeCOL13α and PeCOL13ß, due to an intron-retained AS. The PeCOL13α expressed in the vegetative phase and the reproductive phase, but the PeCOL13ß didn't express during the vegetative phase and showed only a weak expression from F1 to F3 during the reproductive phase. Overexpression of PeCOL13α in rice (Oryza sativa) resulted in a delayed heading time through inhibiting the expressions of Hd3a, OsFTL1, and Ehd1 and activating the expressions of Ghd7 and RCN1. However, the PeCOL13ß-overexpressed rice didn't show any significant differences in flowering compared with wild-type (WT), and the expressions of downstream flowering genes had no notable changes. Further analysis revealed that both PeCOL13α and PeCOL13ß can bind to the PeFT promoter. Meanwhile, PeCOL13α can inhibit the transcription of PeFT, but PeCOL13ß showed no effect. When PeCOL13α and PeCOL13ß coexist, the inhibitory effect of PeCOL13α on PeFT transcription was weakened by PeCOL13ß. This study provides new insights into the mechanism of bamboo flowering research.

SilE-R and SilE-S-DABB Proteins Catalying Enantiospecific Hydrolysis of Organosilyl Ethers.

Silyl ethers fulfil a fundamental role in synthetic organic chemistry as protecting groups and their selective cleavage is an important factor in their application. We present here for the first time two enzymes, SilE-R and SilE-S, which are able to hydrolyse silyl ethers. They belong to the stress-response dimeric A/B barrel domain (DABB) family and are able to cleave the Si-O bond with opposite enantiopreference. Silyl ethers containing aromatic, cyclic or aliphatic alcohols and, depending on the alcohol moiety, silyl functions as large as TBDMS are accepted. The X-ray crystal structure of SilE-R, determined to a resolution of 1.98 Å, in combination with mutational studies, revealed an active site featuring two histidine residues, H8 and H79, which likely act synergistically as nucleophile and Brønsted base in the hydrolytic mechanism, which has not previously been described for enzymes. Although the natural function of SilE-R and SilE-S is unknown, we propose that these 'silyl etherases' may have significant potential for synthetic applications.

Broad Spectrum Enantioselective Amide Bond Synthetase from Streptoalloteichus hindustanus.

The synthesis of amide bonds is one of the most frequently performed reactions in pharmaceutical synthesis, but the requirement for stoichiometric quantities of coupling agents and activated substrates in established methods has prompted interest in biocatalytic alternatives. Amide Bond Synthetases (ABSs) actively catalyze both the ATP-dependent adenylation of carboxylic acid substrates and their subsequent amidation using an amine nucleophile, both within the active site of the enzyme, enabling the use of only a small excess of the amine partner. We have assessed the ability of an ABS from Streptoalloteichus hindustanus (ShABS) to couple a range of carboxylic acid substrates and amines to form amine products. ShABS displayed superior activity to a previously studied ABS, McbA, and a remarkable complementary substrate specificity that included the enantioselective formation of a library of amides from racemic acid and amine coupling partners. The X-ray crystallographic structure of ShABS has permitted mutational mapping of the carboxylic acid and amine binding sites, revealing key roles for L207 and F246 in determining the enantioselectivity of the enzyme with respect to chiral acid and amine substrates. ShABS was applied to the synthesis of pharmaceutical amides, including ilepcimide, lazabemide, trimethobenzamide, and cinepazide, the last with 99% conversion and 95% isolated yield. These findings provide a blueprint for enabling a contemporary pharmaceutical synthesis of one of the most significant classes of small molecule drugs using biocatalysis.

RNF112-mediated FOXM1 ubiquitination suppresses the proliferation and invasion of gastric cancer.

Forkhead box M1 (FOXM1) plays a critical role in development physiologically and tumorigenesis pathologically. However, insufficient efforts have been dedicated to exploring the regulation, in particular the degradation of FOXM1. Here, the ON-TARGETplus siRNA library targeting E3 ligases was used to screen potential candidates to repress FOXM1. Of note, mechanism study revealed that RNF112 directly ubiquitinates FOXM1 in gastric cancer, resulting in a decreased FOXM1 transcriptional network and suppressing the proliferation and invasion of gastric cancer. Interestingly, the well-established small-molecule compound RCM-1 significantly enhanced the interaction between RNF112 and FOXM1, which further promoted FOXM1 ubiquitination and subsequently exerted promising anticancer effects in vitro and in vivo. Altogether, we demonstrate that RNF112 suppresses gastric cancer progression by ubiquitinating FOXM1 and highlight the RNF112/FOXM1 axis serves as both prognosis biomarker and therapeutic target in gastric cancer.

Increasing the yield of drip-irrigated rice by improving photosynthetic performance and enhancing nitrogen metabolism through optimizing water and nitrogen management.

Drip irrigation under plastic film mulching is an important technique to achieve water-conserving and high-efficiency rice (Oryza sativa L.) production in arid areas, but the grain yield of drip-irrigated rice is much lower than the expected yield (10.9-12.05 t·hm-2) in practical production applications. Therefore, we hope to further understand the photosynthetic physiological mechanism of drip-irrigated rice yield formation by optimizing water and nitrogen management during the growth period and provide a scientific reference for improving yield and nitrogen use efficiency (NUE) of drip-irrigated rice in arid areas. In 2020 and 2021, T-43 (a drought-resistant; V1) and Liangxiang-3 (a drought-sensitive cultivar; V2) were cultivated under two water treatments (W1: limited drip irrigation, 10200 m3·hm-2; W2: deficit drip irrigation, 8670 m3·hm-2) and three nitrogen fertilization modes with different ratios of seedling fertilizer:tillering fertilizer:panicle fertilizer:grain fertilizer (N1, 30%:50%:13%:7%; N2, 20%:40%:30%:10%; and N3, 10%:30%:40%:20%). The photosynthetic characteristics, nitrogen metabolism, yield, and NUE were analysed. The results showed that compared with other treatments, the W1N2 resulted in 153.4-930.3% higher glutamate dehydrogenase (GDH) contents and 19.2-49.7% higher net photosynthetic rates (P n) in the leaves of the two cultivars at 20 days after heading, as well as higher yields and NUE. The two cultivars showed no significant difference in the physiological changes at the panicle initiation stage, but the P n, abscisic acid (ABA), indole acetic acid (IAA), gibberellic acid (GA3), and zeatin riboside (ZR) levels of V1 were higher than those of V2 by 53.1, 25.1, 21.1, 46.3 and 36.8%, respectively, at 20 days after heading. Hence, V1 had a higher yield and NUE than V2. Principal component analysis revealed that P n and GDH were the most important physiological factors affecting rice yield performance. In summary, the W1N2 treatment simultaneously improved the yield and NUE of the drought-resistant rice cultivar (T-43) by enhancing the photosynthetic characteristics and nitrogen transport capacity and coordinating the balance of endogenous hormones (ABA, IAA, GA3, and ZR) in the leaves.

A Classification Method of Point Clouds of Transmission Line Corridor Based on Improved Random Forest and Multi-Scale Features.

Classification of airborne laser scanning (ALS) point clouds of power lines is of great importance to their reconstruction. However, it is still a difficult task to efficiently and accurately classify the ground, vegetation, power lines and power pylons from ALS point clouds. Therefore, in this paper, a method is proposed to improve the accuracy and efficiency of the classification of point clouds of transmission lines, which is based on improved Random Forest and multi-scale features. The point clouds are filtered by the optimized progressive TIN densification filtering algorithm, then the elevations of the filtered point cloud are normalized. The features of the point cloud at different scales are calculated according to the basic features of the point cloud and the characteristics of transmission lines. The Relief F and Sequential Backward Selection algorithm are used to select the best subset of features to estimate the parameters of the learning model, then an Improved Random Forest classification model is built to classify the point clouds. The proposed method is verified by using three different samples from the study area and the results show that, compared with the methods based on Support Vector Machines, AdaBoost or Random Forest, our method can reduce feature redundancy and has higher classification accuracy and efficiency.

Biotechnology in Future Food Lipids: Opportunities and Challenges.

Lipids are a large group of essential nutrients in daily diets that provide energy and maintain various physiological functions. As the global population is rapidly expanding, there is an urgent need to enhance the production and quality of food lipids. The development of modern biotechnology allows the manipulation of oil production in plants and microorganisms and the improvement of the nutritional value of food lipids. Various metabolic engineering strategies have been exploited to increase oil production and produce value-added oils in traditional oil crops and other novel lipid sources (e.g., plant vegetative tissues, microalgae, and oleaginous microorganisms). Furthermore, natural lipid structures can be modified by lipases to prepare functional lipids, e.g., diacylglycerols, medium-long-medium-type structured triacylglycerols, human milk-fat substitutes, and structuralphospholipids, for specific nutritional demands. In this review, we focus on the recent advances in metabolic engineering of lipid production in plants and microorganisms, and the preparation of functional lipids via biocatalysis.

FOXM1 increases hTERT protein stability and indicates poor prognosis in gastric cancer.

Gastric cancer is one of most lethal diseases across the world. However, the underlying mechanism of gastric cancer carcinogenesis and development is still not fully known. Forkhead box M1 (FOXM1) belongs to the FOX family and has crucial roles in transactivation of multiple oncogenes in several cancer types, including gastric cancer. Recent studies have also shown the non-transcriptional function of FOXM1 via protein-protein interactions. Human telomerase reverse transcriptase (hTERT) is the core subunit of telomerase that facilitates cancer initiation and progression by maintaining cell immortalization, promoting cell proliferation and inhibiting cell apoptosis. However, the relationship between FOXM1 and hTERT in gastric cancer is still unclear. In our study, we found that FOXM1 and hTERT were convergent to the cell cycle-related pathways and they were positively related with advanced gastric cancer stages and poor outcomes. Simultaneous high levels of FOXM1 and hTERT predicted the worst prognosis. FOXM1 could increase hTERT protein rather than mRNA levels in a non-transcriptional manner. Mechanistically, FOXM1 interrupted the interaction between the E3 ligase MKRN1 and hTERT and decreased hTERT protein degradation. Further studies revealed that FOXM1 interacted with hTERT through its DNA-binding domain (DBD) region. Finally, we found that hTERT played important roles in FOXM1-mediated activation of the Wnt/ß-catenin pathway to promote gastric cancer cell proliferation. Taken together, we found a novel non-classical function of FOXM1 to increase hTERT protein stability. Targeting the FOXM1-hTERT pathway may be a potential therapeutic strategy in treating gastric cancer.

Enzymatic Photometric Assays for the Selective Detection of Halides.

Halides are substrates and products of a number of biotechnologically important enzymes like dehalogenases, halide methyltransferases, and halogenases. Therefore, the determination of halide concentrations in samples is important. The classical methods based on mercuric thiocyanate are very dangerous, produce hazardous waste, and do not discriminate between chloride, bromide, and iodide. In this chapter, we describe a detailed protocol for the determination of halide concentrations based on the haloperoxidase-catalyzed oxidation of halides. The resulting hypohalous acids are detected using commercially available colorimetric or fluorimetric probes. The biocatalytic nature of the assays allows them to be implemented in one-pot cascade reactions with halide-generating enzymes. Since chloride is ubiquitous in biological systems, we also describe convenient photometric assays for the selective detection of bromide and iodide in the presence of chloride, obviating the need for laborious dialyses to obtain halide-free enzymes and reagents.

Metalloid-Cluster Ligands Enabling Stable and Active FeN4 -Ten Motifs for the Oxygen Reduction Reaction.

In nature, the oxygen reduction reaction (ORR) is catalyzed by cytochrome P450 (CYP) enzymes containing heme iron centers with an axial thiolate ligand (FeN4 -S), which are among the most finely developed catalysts by natural selection. However, the exceptional ORR activity and selectivity of CYP enzymes originate from their non-rigid and self-adaptive coordination network with molecular ligands, which sacrifices the stability of the active motifs under electrochemical reaction conditions. Here, a design strategy to circumvent this dilemma by incorporating Fe-N4 motifs into carbon matrices instead of the protein scaffold and replacing the axial molecular thiolate ligand with a stable tellurium cluster (Ten ) is demonstrated. Theoretical calculations indicate a moderate interaction between Fe 3d and Te 5p orbitals once n > 2, allowing the FeTe bond to dynamically change its strength to adaptively facilitate the intermediate steps during the ORR process, which renders FeN4 -Ten active sites with superior ORR activity. This adaptive behavior mimics the conformational dynamics of an enzyme during the reaction, but retains the stability nature as a heterogeneous catalyst. The experiments validate that the as-designed catalyst with a characterized FeN4 -Ten structure outperforms the commercial Pt/C catalyst both on activity and stability.

Correction: Tang et al. Can El Niño-Southern Oscillation Increase Respiratory Infectious Diseases in China? An Empirical Study of 31 Provinces. Int. J. Environ. Res. Public Health 2022, 19, 2971.

In the original publication [...].

Can El Niño-Southern Oscillation Increase Respiratory Infectious Diseases in China? An Empirical Study of 31 Provinces.

Respiratory infectious diseases (RID) are the major form of infectious diseases in China, and are highly susceptible to climatic conditions. Current research mainly focuses on the impact of weather on RID, but there is a lack of research on the effect of El Niño-Southern Oscillation (ENSO) on RID. Therefore, this paper uses the system generalized method of moments (SYS-GMM) and the data of 31 provinces in China from 2007 to 2018 to construct a dynamic panel model to empirically test the causality between ENSO and RID morbidity. Moreover, this paper considers the moderating effects of per capita disposable income and average years of education on this causality. The results show that ENSO can positively and significantly impact RID morbidity, which is 5.842% higher during El Niño years than normal years. In addition, per capita disposable income and average years of education can effectively weaken the relationship between ENSO and RID morbidity. Thus, this paper is of great significance for improving the RID early climate warning system in China and effectively controlling the spread of RID.

Association Between Nutritional Risk Screening Score and Prognosis of Patients with Sepsis.

BACKGROUND: Malnutrition is one of the most critical factors affecting patients' risk of infection and length of stay, and it may affect the prognosis of patients with sepsis. There have been no studies that have applied nutritional risk screening tools to stratify patients with sepsis according to prognosis. METHODS: We retrospectively analyzed the clinical data of 425 adult sepsis inpatients who were grouped based on nutritional risk screening (NRS) score, including a nutrition score, disease severity score, and age score. Prognostic factors were analyzed using univariate and multivariate regression analyses. RESULTS: Of the enrolled patients, 174 had an NRS score of ≥3; these patients were older and had a longer hospitalization time but lower body mass index (BMI), albumin (ALB) than others. Univariate Cox regression analysis showed that age, ALB, C-reactive protein (CRP), and NRS score were significantly (P<0.05) associated with in-hospital mortality. Multivariate analysis showed that age (hazard ratio [HR]=1.020, 95% confidence interval [CI]: 1.005-1.036; P=0.008) and ALB (HR=0.924, 95% CI: 0.885-0.966; P<0.001) were independent risk factors for sepsis-related mortality. The Kaplan-Meier analysis revealed that the cumulative in-hospital mortality of sepsis patients with an NRS score of ≥3 was significantly higher than that of patients with an NRS score of <3 (P=0.022). CONCLUSION: NRS scores can effectively risk stratify sepsis patients. Patients with high NRS scores should be monitored more closely to halt further disease progression.

Enhancement of Phospholipid Binding and Catalytic Efficiency of Streptomyces klenkii Phospholipase D by Increasing Hydrophobicity of the Active Site Loop.

The mechanism of active site loops of Streptomyces phospholipase D (PLD) binding to the lipid-water interface for catalytic reactions still remains elusive. A flexible loop (residues 376-382) in the active site of Streptomyces klenkii PLD (SkPLD) is conserved within PLDs in most of the Streptomyces species. The residue Ser380 was found to be essential for the enzyme's adsorption to the interface and its substrate recognition. The S380V mutant showed a 4.8 times higher catalytic efficiency and nearly seven times higher adsorption equilibrium coefficient compared to the wild-type SkPLD. The monolayer film technique has confirmed that the substitution of Ser380 with valine in the loop exhibited positive interaction between the enzyme and PCs with different acyl chain lengths. The results of the interfacial binding properties indicated that the S380V mutant might display suitable phosphatidylserine synthesis activity. The present study will be helpful to explain the role of residue 380 in the active site loops of Streptomyces PLD.

From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases.

Halide methyltransferases (HMTs) enable the enzymatic synthesis of S-adenosyl-l-methionine (SAM) from S-adenosyl-l-homocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemo- and regioselective biocatalytic alkylations.

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs.

Biocatalytic alkylations are important reactions to obtain chemo-, regio- and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4-dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

CircMRPS35 suppresses gastric cancer progression via recruiting KAT7 to govern histone modification.

BACKGROUND: Aberrant expression of circular RNAs contributes to the initiation and progression of cancers, but the underlying mechanism remains elusive. METHODS: RNA-seq and qRT-PCR were performed to screen differential expressed circRNAs between gastric cancer tissues and adjacent normal tissues. Candidate circRNA (circMRPS35) was screened out and validated by qRT-PCR. Cell proliferation and invasion ability were determined by CCK-8 and cell invasion assays. RNA-seq, GO-pathway, RNA pull-down and ChIRP were further applied to search for detailed mechanism. RESULTS: Here, a novel circRNA named circMRPS35, was screened out by RNA-seq in gastric cancer tissues, whose expression is related to clinicopathological characteristics and prognosis in gastric cancer patients. Biologically, circMRPS35 suppresses the proliferation and invasion of gastric cancer cells in vitro and in vivo. Mechanistically, circMRPS35 acts as a modular scaffold to recruit histone acetyltransferase KAT7 to the promoters of FOXO1 and FOXO3a genes, which elicits acetylation of H4K5 in their promoters. Particularly, circMRPS35 specifically binds to FOXO1/3a promoter regions directly. Thus, it dramatically activates the transcription of FOXO1/3a and triggers subsequent response of their downstream target genes expression, including p21, p27, Twist1 and E-cadherin, resulting in the inhibition of cell proliferation and invasion. Moreover, circMRPS35 expression positively correlates with that of FOXO1/3a in gastric cancer tissues. CONCLUSIONS: Our findings not only reveal the pivotal roles of circMRPS35 in governing histone modification in anticancer treatment, but also advocate for triggering circMRPS35/KAT7/FOXO1/3a pathway to combat gastric cancer.

Improving the Catalytic Activity and Thermostability of MAS1 Lipase by Alanine Substitution.

MAS1 is a lipase isolated from Streptomyces sp. strain W007 with potential application in biotechnology. Structural analysis of MAS1 lipase showed that eight amino acids with bulkier side located in the substrate-binding pocket may be involved in affecting catalytic performance. Alanine substitutions of those residues were conducted to reduce steric clash of catalyzed pocket and probe their functional roles. The kcat/Km of mutants H108A, F153A, and V233A increased to 2.3-, 2.1-, and 1.4-fold, respectively. Interestingly, the half-life (60 °C) of F153A had shifted to 523 min after mutagenesis, which was fivefold enhancement toward that of MAS1 wide-type. Furthermore, higher hydrolysis ability of mutants H108A and F153A toward palm stearin of high melting temperature made them potentially applicable in oil/fat modification. Our work provided an example to obtain biocatalysts with desired catalytic behaviors by protein engineering.

The Role of Residues 103, 104, and 278 in the Activity of SMG1 Lipase from Malassezia globosa: A Site-Directed Mutagenesis Study.

The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Sitedirected mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and pnitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipidanchoring mechanism for DAG (L103 and F104 serve as "anchors" to the lipid interface) were proposed.