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Abstract Gangliosides play vital biological regulatory roles and are associated with neurological system diseases, malignancies, and immune deficiencies. They have received extensive attention in developing targeted drugs and diagnostic markers. However, it is difficult to obtain enough structurally defined gangliosides and analogs especially at an industrial-relevant scale, which prevent exploring structure-activity relationships and identifying drug ingredients. Here, we report a highly modular chemoenzymatic cascade assembly (MOCECA) strategy for customized and large-scale synthesis of ganglioside analogs with various glycan and ceramide epitopes. We typically accessed five gangliosides with therapeutic promising and systematically prepared ten GM1 analogs with diverse ceramides. Through further process amplification, we achieved industrial production of ganglioside GM1 in the form of modular assembly at hectogram scale. Using MOCECA-synthesized GM1 analogs, we found unique ceramide modifications on GM1 could enhance the ability to promote neurite outgrowth. By comparing the structures with synthetic analogs, we further resolved the problem of contradicting descriptions for GM1 components in different pharmaceutical documents by reinterpreting the exact two-component structures of commercialized GM1 drugs. Because of its applicability and stability, the MOCECA strategy can be extended to prepare other glycosphingolipid structures, which may pave the way for developing new glycolipid drugs.

Abstract Background Since Nicotiana tabacum (tobacco) has important significance to humans for their medicinal uses, to find antivirus activities inhibitors from tobacco, increase its medicinal value, and comprehensive utilization of its by-products, our group had investigated the chemical constituents of the stems of Y-202, a cultivar of tobacco which high resistance to tobacco mosaic virus (TMV). Results Four new isoindolinone-type alkaloids, nicoisoindoles A–D (1–4), along with four known isoindole derivatives (5–8) were isolated. Compounds 1–4 represent a new subclass of isoindolinone alkaloids with rare cyclopenta[f]isoindole-1-one frameworks. Among them, nicoisoindole C (3) possesses an unusual N-2-(5-methoxy-6-methylpyridin-2-yl) ethyl moiety, while nicoisoindole D (4) has a novel a N-(3-methyl-6-oxo-1,6-dihydropyridin-2-yl)methyl substituent. Interestingly, compounds 1, 3, and 4 showed high anti-TMV activities with inhibition rates of 43.8%, 58.8%, and 67.8% at the concentration of 20 μM, and IC50 values of 23.6, 19.5 and 15.4 μM, respectively, even more potent than that of positive control. Conclusions The successful isolation and structure identification of new oxocyclopenta[f]isoindole-1-ones provide materials for the screening of antivirus activities inhibitors, and contribute to the development and utilization of the waste from tobacco cultivation. Graphical Abstract

Abstract In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography methods are labor intensive, time consuming and not compatible with real-time analysis. Here, we developed an inexpensive, high-throughput, and real-time detection method for the monitoring of in vitro RNA synthesis called iSpinach aptamer-based monitoring of Transcription Activity in Real-time (STAR). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the green fluorescent protein gene and the 3T4T variants demonstrated significantly enhanced transcription output, with at least 1.7-fold and 2.8-fold greater output than the wild-type DNA template and common transcription template, respectively. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA-related enzymes and new drugs. Graphical Abstract iSpinach aptamer-based monitoring of transcription activity in real-time (STAR).

Colorectal cancer (CRC) is the third leading cause of cancer mortality in the United States, with an estimated 52,000 deaths in 2023. Though significant progress has been made in both diagnosis and treatment of CRC in recent years, genetic heterogeneity of CRC—the culprit for possible CRC relapse and drug resistance, is still an insurmountable challenge. Thus, developing more effective therapeutics to overcome this challenge in new CRC treatment strategies is imperative. Genetic and epigenetic changes are well recognized to be responsible for the stepwise development of CRC malignancy. In this review, we focus on detailed genetic alteration information about the nuclear factor (NF)-κB signaling, including both NF-κB family members, and their regulators, such as protein arginine methyltransferase 5 (PRMT5), and outer dynein arm docking complex subunit 2 (ODAD2, also named armadillo repeat-containing 4, ARMC4), etc., in CRC patients. Moreover, we provide deep insight into different CRC research models, with a particular focus on patient-derived xenografts (PDX) and organoid models, and their potential applications in CRC research. Genetic alterations on NF-κB signaling components are estimated to be more than 50% of the overall genetic changes identified in CRC patients collected by cBioportal for Cancer Genomics; thus, emphasizing its paramount importance in CRC progression. Consequently, various genetic alterations on NF-κB signaling may hold great promise for novel therapeutic development in CRC. Future endeavors may focus on utilizing CRC models (e.g., PDX or organoids, or isogenic human embryonic stem cell (hESC)-derived colonic cells, or human pluripotent stem cells (hPSC)-derived colonic organoids, etc.) to further uncover the underpinning mechanism of these genetic alterations in NF-κB signaling in CRC progression. Moreover, establishing platforms for drug discovery in dishes, and developing Biobanks, etc., may further pave the way for the development of innovative personalized medicine to treat CRC in the future.

UDP-Glycosyltransferases (UGTs) catalyze the transfer of nucleotide-activated sugars to specific acceptors, among which the GT1 family enzymes are well-known for their function in biosynthesis of natural product glycosides. Elucidating GT function represents necessary step in metabolic engineering of aglycone glycosylation to produce drug leads, cosmetics, nutrients and sweeteners. In this review, we systematically summarize the phylogenetic distribution and catalytic diversity of plant GTs. We also discuss recent progress in the identification of novel GT candidates for synthesis of plant natural products (PNPs) using multi-omics technology and deep learning predicted models. We also highlight recent advances in rational design and directed evolution engineering strategies for new or improved GT functions. Finally, we cover recent breakthroughs in the application of GTs for microbial biosynthesis of some representative glycosylated PNPs, including flavonoid glycosides (fisetin 3-O-glycosides, astragalin, scutellarein 7-O-glucoside), terpenoid glycosides (rebaudioside A, ginsenosides) and polyketide glycosides (salidroside, polydatin).

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory condition of the esophagus associated with elevated esophageal eosinophils. Second only to gastroesophageal reflux disease, EoE is one of the leading causes of chronic refractory dysphagia in adults and children. EoE is a clinicopathologic disorder and the histological portion of the diagnosis requires enumerating the density of esophageal eosinophils in esophageal biopsies, and evaluating additional features such as basal zone hyperplasia is helpful. However, this task requires time-consuming, somewhat subjective manual analysis, thus reducing the ability to process the complex tissue structure and infer its relationship with the patient's clinical status. Previous artificial intelligence (AI) approaches that aimed to improve histology-based diagnosis focused on recapitulating identification and quantification of the area of maximal eosinophil density, the gold standard manual metric for determining EoE disease activity. However, this metric does not account for the distribution of eosinophils or other histological features, over the whole slide image. Here, we developed an artificial intelligence platform that infers local and spatial biomarkers based on semantic segmentation of intact eosinophils and basal zone distributions. Besides the maximal density of eosinophils [referred to as Peak Eosinophil Count (PEC)] and a maximal basal zone fraction, we identify the value of two additional metrics that reflect the distribution of eosinophils and basal zone fractions. This approach enables a decision support system that predicts EoE activity and potentially classifies the histological severity of EoE patients. We utilized a cohort that includes 1,066 biopsy slides from 400 subjects to validate the system's performance and achieved a histological severity classification accuracy of 86.70%, sensitivity of 84.50%, and specificity of 90.09%. Our approach highlights the importance of systematically analyzing the distribution of biopsy features over the entire slide and paves the way toward a personalized decision support system that will assist not only in counting cells but can also potentially improve diagnosis and provide treatment prediction.

Background: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. Methods: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. Findings: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. Interpretation: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. Fund: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117. Keywords: Advanced hepatocellular carcinoma, T-type voltage gated calcium channels, Calcium influx, Cav 3·2, CACNA1H, Amplitude-modulated, Radiofrequency, Electromagnetic fields, AM RF EMF

A piecewise fast multi-power reaching law (PFMPRL) is proposed aiming at the problems of chattering and slow convergence in the reaching phase of sliding mode control (SMC). In this paper, the fast power reaching law and the double power reaching law are combined, and a nonlinear function is introduced to design the exponential term in PFMPRL. The proposed method ensures the characteristic of fast convergence of the system at all the phases of tendency. The characteristic of fixed-time convergence has also been satisfied. The study proves that the system state can converge to steady-state error bounds within a finite time in the presence of system uncertainty and bounded external disturbance. Compared with the existed methods, the proposed method has shorter convergence time and smaller steady-state error bound. To suppress the influence of model uncertainty and disturbance in system control, a non-linear disturbance observer (NDO) is introduced, and combined with the reaching law-based non-singular terminal sliding mode control (NTSMC), is applied to the cart inverted pendulum system. Simulation results and numerical analysis verify the effectiveness and superiority of this approach.

Abstract Background Lipoxygenase (LOX) from Anabaena sp. PCC 7120 (Ana-rLOX) offers important applications in the food industry, especially for improving aroma and dough rheological properties. However, industrial applications of LOXs have been limited by their poor thermostability. Herein, we report a bioinformatics-based consensus concept approach for the engineering of thermostable Ana-rLOX. Results A series of mutations (N130D, G260A, S437T, N130D/G260Q, N130D/S437Y) showed higher thermostability and activity than the wild-type enzyme. Thus, N130D/G260Q exhibited a 6.6-fold increase in half-life and 2.45 °C increase in unfolding temperature; N130D/S437Y showed a 10 °C increase in optimal temperature. The secondary structure did not change much that contributed to improved thermostability were investigated in detail using circular dichroism. Homology modeling suggested that enhanced thermostability and specific activity may result from favorable hydrophobic interactions. Conclusions A series of mutations were achieved, showing higher thermostability and activity than the wild-type enzyme by semi-rational mutagenesis with limited structure information. Our findings provide important new insights into molecular modifications aimed at improving Ana-rLOX thermostability and activity.

Optimizing an enzyme usually requires testing thousands of variants, thus consuming large amounts of material and time. Here, the authors present a method that allows for measuring two different activities of the same enzyme simultaneously instead of doing two consecutive rounds of screening.

Polyunsaturated fatty acids (PUFAs) including epoxide-modified ω-3 and ω-6 fatty acids are made via oxidation to create highly polarized carbon-oxygen bonds crucial to their function as signaling molecules. A critical PUFA, arachidonic acid (ARA), is metabolized to a diverse set of lipids signaling molecules through cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 epoxygenase, or cytochrome P450 hydroxylase; however, the majority of ARA is metabolized into anti-inflammatory epoxides via cytochrome P450 enzymes. These short-lived epoxide lipids are rapidly metabolized or inactivated by the soluble epoxide hydrolase (sEH) into diol-containing products. sEH inhibition or knockout has been a practical approach to study the biology of the epoxide lipids, and has been shown to effectively treat inflammatory conditions in the preclinical models including gastrointestinal ulcers and colitis by shifting oxylipins to epoxide profiles, inhibiting inflammatory cell infiltration and activation, and enhancing epithelial cell defense via increased mucin production, thus providing further evidence for the role of sEH as a pro-inflammatory protein. Non-steroidal anti-inflammatory drugs (NSAIDs) with COX-inhibitor activity are among the most commonly used analgesics and have demonstrated applications in the management of cardiovascular disease and intriguingly cancer. Major side effects of NSAIDs however are gastrointestinal ulcers which frequently precludes their long-term application. In this review, we hope to bridge the gap between NSAID toxicity and sEH-mediated metabolic pathways to focus on the role of epoxy fatty acid metabolic pathway of PUFAs in NSAIDS-ulcer formation and healing as well as inflammation-related carcinogenesis. Specifically we address the potential application of sEH inhibition to enhance ulcer healing at the site of inflammation via their activity on altered lipid signaling, mitochondrial function, and diminished reactive oxygen species, and further discuss the significance of dual COX and sEH inhibitor in anti-inflammation and carcinogenesis.

Shift work and jet lag, characterized by circadian misalignment, can disrupt several physiological activities, but whether they affect the rhythm of glucose uptake and insulin sensitivity remain unclear. In the present study, female C57BL/6J mice were maintained for four weeks under the condition of 8-hour phase advance and delay every 3-4 days to mimic shift work. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed repeatedly at Zeitgeber time (ZT) 0, ZT6, ZT12, and ZT18. Glucose-stimulated insulin secretion (GSIS) test was performed at ZT6. We found that the average level of daily glucose tolerance did not decrease but the phase of glucose tolerance advanced by 2.27 hours and the amplitude attenuated by 20.4% in shift work mice. At ZT6, IPITT showed blood glucose at 30 min after insulin injection decreased faster in shift work mice (-3.50±0.74mmol/L, -61.58±7.89%) than that in control mice (-2.11±1.10mmol/L, -33.72±17.24%), but IPGTT and GSIS test showed no significant difference between the two groups. Food intake monitor showed that the feeding time of shift work mice continued to advance. Restricting feed to a fixed 12-hour period alleviated the increase of insulin sensitivity induced by shift-work. We also observed that an increase of blood glucose and liver glycogen at ZT0, as well as a phase advance of liver clock genes and some glucose metabolism-related genes such as forkhead box O1 (Foxo1) and peroxisome proliferator activated receptor alpha (Pparα) in shift work mice. Our results showed that light change-simulated shift work altered insulin sensitivity during the light phase and shifted glucose tolerance rhythms in female mice, suggesting a causal association between long-term shift work and type 2 diabetes.

Cell-free synthetic biology system organizes multiple enzymes (parts) from different sources to implement unnatural catalytic functions. Highly adaption between the catalytic parts is crucial for building up efficient artificial biosynthetic systems. Protein engineering is a powerful technology to tailor various enzymatic properties including catalytic efficiency, substrate specificity, temperature adaptation and even achieve new catalytic functions. However, altering enzymatic pH optimum still remains a challenging task. In this study, we proposed a novel sequence homolog-based protein engineering strategy for shifting the enzymatic pH optimum based on statistical analyses of sequence-function relationship data of enzyme family. By two statistical procedures, artificial neural networks (ANNs) and least absolute shrinkage and selection operator (Lasso), five amino acids in GH11 xylanase family were identified to be related to the evolution of enzymatic pH optimum. Site-directed mutagenesis of a thermophilic xylanase from Caldicellulosiruptor bescii revealed that four out of five mutations could alter the enzymatic pH optima toward acidic condition without compromising the catalytic activity and thermostability. Combination of the positive mutants resulted in the best mutant M31 that decreased its pH optimum for 1.5 units and showed increased catalytic activity at pH

Eosinophilic gastrointestinal disorders (EGID) are characterized pathologically by excess eosinophils in mucosal biopsies of one or multiple sites in the gastrointestinal (GI) tract, simultaneously or sequentially. Eosinophilic esophagitis (EoE) is the best characterized EGID, and in most patients it is an abnormal immune-mediated response to food antigens. Current recommendations for diagnosis include signs and symptoms of esophageal dysfunction that do not respond to proton-pump inhibitor therapy, and esophageal biopsies that exhibit at least 15 intraepithelial eosinophils in at least one high power field (HPF). Therapy consists of swallowed glucocorticoids or dietary elimination. Eosinophilic gastritis (EG) is the second most common form of EGID, but like all forms of EGID except EoE consensus recommendations for either clinical or pathological diagnosis do not exist. EG may be associated clinically with peripheral blood eosinophilia, hypoalbuminemia, and anemia, and pathologically with marked expansion of lamina propria by dense eosinophilic infiltrates. Eosinophilic enteritis (EE) may be subdivided into eosinophilic duodenitis, eosinophilic jejunitis, and eosinophilic ileitis. Most investigators believe that EE rarely, if ever, exists as a solitary form of EGID and is encountered only in patients who have at least one other affected portion of the GI tract. Eosinophilic colitis (EC) is perhaps the most enigmatic EGID. Distinction of EC from inflammatory bowel disease may be problematic especially in children. Multiple possible etiologies for EGID include hypereosinophilic syndrome, drug reactions, etc. Currently, the only etiology that can be identified histologically is parasitic infestation, if a portion of an invasive parasite is found in mucosal biopsies. This review will provide guidelines for the pathologic diagnosis of the various forms of EGID.

Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca2+ and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473) and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.

Merkel cell carcinoma (MCC) is an uncommon cutaneous malignant tumor that presents as a rapidly growing skin nodule on sun-exposed areas of the body. MCC is aggressive with regional nodal and distant metastases to the skin, lung, and bones. There have been no reports of metastatic MCC to the mesentery and 6 reports describing metastasis to the small intestine. We present a case of metastatic MCC to the mesentery with infiltration to the small bowel, 8 years after original tumor resection. This is the 5th metastasis and it encased the small bowel resulting in a hair-pin loop contributing to the unusual clinical presentation. Although MCC metastatic to the bowel is uncommon, it is not rare. It is important to recognize the unusual manifestations of this disease as they are becoming more common in the future. Routine radiologic surveillance and thorough review of systems are important to patient follow-up.