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BackgroundResearch of immunotherapy for cholangiocarcinoma has yielded some results, but more clinical data are needed to prove its efficacy and safety. Moreover, there is a need to identify accessible indexes for selecting patients who may benefit from such treatments.MethodsThe medical records of 66 cholangiocarcinoma patients who underwent immunotherapy were retrospectively collected. The effectiveness of immunotherapy was assessed by tumor response, progression-free survival (PFS), and overall survival (OS), while safety was evaluated by adverse events during treatment. Univariate and multivariate Cox regression analyses were performed to identify prognostic risk factors for PFS and OS, and Kaplan-Meier curves of potential prognostic factors were drawn.ResultsOverall, in this study, immunotherapy achieved an objective response rate of 24.2% and a disease control rate of 89.4% for the included patients. The median PFS was 445 days, and the median OS was 772.5 days. Of the 66 patients, 65 experienced adverse events during treatment, but none had severe consequences. Multivariate Cox analysis indicated that tumor number is a prognostic risk factor for disease progression following immunotherapy in cholangiocarcinoma patients, while tumor differentiation and the fibrosis-4 (FIB-4) index are independent risk factors for OS.ConclusionIn general, immunotherapy for cholangiocarcinoma is safe, with adverse events remaining within manageable limits, and it can effectively control disease progression in most patients. The FIB-4 index may reflect the potential benefit of immunotherapy for patients with cholangiocarcinoma.

Hepatoid carcinoma is an extrahepatic primary tumor displaying characteristics reminiscent of hepatocellular carcinoma differentiation, which is found in various organs, such as the stomach, ovaries, gallbladder, and pancreas. Reports of pancreatic hepatoid carcinoma remain scarce. Consequently, understanding of this disease remains a priority, with no established consensus on its diagnosis and management. Here, we reported the case of a 45-year-old woman diagnosed with hepatoid carcinoma located in the pancreatic head, accompanied by multiple lymph node metastases. Following pancreaticoduodenectomy, the patient developed liver metastases within 3 months. Subsequently, she underwent adjuvant therapy consisting of Teysuno and Durvalumab following microwave ablation for the liver metastases. Remarkably, the patient has survived for one year without significant disease progression. This case underscores the potential efficacy of immunotherapy as a promising treatment option for pancreatic hepatoid carcinoma. Further research and clinical trials are warranted to explore the optimal management strategies for this rare and challenging malignancy.

BackgroundEctopic lipid deposition plays a promoting role in many chronic metabolic diseases. Abnormal adipogenic differentiation of mesenchymal stem cells (MSCs) is an important cause of lipid deposition in organs. Studies have shown that serum angiopoietin-like protein 8 (ANGPTL8) levels are increased in patients with many chronic metabolic diseases (such as type 2 diabetes, obesity, and hepatic steatosis), while the role of ANGPTL8 in ectopic lipid accumulation has not been reported.MethodsWe used the Gene Expression Omnibus (GEO) database to analyze the expression of ANGPTL8 in subcutaneous adipose tissue of obese patients and qPCR to analyze the expression of ANGPTL8 in the liver of high-fat diet (HFD)-induced obese mice. To explore the potential roles of ANGPTL8 in the progression of ectopic lipid deposition, ANGPTL8 knockout (KO) mice were constructed, and obesity models were induced by diet and ovariectomy (OVX). We analyzed lipid deposition (TG) in the liver, kidney, and heart tissues of different groups of mice by Oil Red O, Sudan black B staining, and the single reagent GPO-PAP method. We isolated and characterized MSCs to analyze the regulatory effect of ANGPTL8 on Wnt/β-Catenin, a key pathway in adipogenic differentiation. Finally, we used the pathway activator LiCl and a GSK3β inhibitor (i.e., CHIR99021) to analyze the regulatory mechanism of this pathway by ANGPTL8.ResultsANGPTL8 is highly expressed in the subcutaneous adipose tissue of obese patients and the liver of HFD-induced obese mice. Both normal chow diet (NCD)- and HFD-treated ANGPTL8 KO male mice gained significantly less weight than wild-type (WT) male mice and reduced ectopic lipid deposition in organs. However, the female mice of ANGPTL8 KO, especially the HFD group, did not show differences in body weight or ectopic lipid deposition because HFD could induce estrogen overexpression and then downregulate ANGPTL8 expression, thereby counteracting the reduction in HFD-induced ectopic lipid deposition by ANGPTL8 deletion, and this result was also further proven by the OVX model. Mechanistic studies demonstrated that ANGPTL8 could promote the differentiation of MSCs into adipocytes by inhibiting the Wnt/β-Catenin pathway and upregulating PPARγ and c/EBPα mRNA expression.ConclusionsANGPTL8 promotes the differentiation of MSCs into adipocytes, suggesting that ANGPTL8 may be a new target for the prevention and treatment of ectopic lipid deposition in males.

Cinnamic acid (CA) is a safe and effective antimicrobial agent. The objective of this study was to reveal the antibacterial mechanism of CA against a food-derived Pseudomonas fragi 38-8, from the aspects of bacterial growth kinetics, cell membrane homeostasis, cell microstructure, and transcription. The minimum inhibitory concentration (MIC) of CA against P. fragi 38-8 was 0.25 mg/ml. CA retarded bacterial growth and induced a series of cell membrane changes. After CA treatment, cell membrane homeostasis was destroyed, which was evidenced by cell membrane depolarization, intracellular pH reduction, and intracellular ATPase activity decrease. Field emission scanning electron microscope (FESEM), transmission electron microscope (TEM), and confocal laser scanning fluorescence microscope (CLSM) realized the visualization of cell microstructure changes, showing cell death and morphological changes, such as cell rupture, shrinkage, and hollowness. RNA sequencing analysis further confirmed the effects of CA to the cell membrane, because of the significant enrichment of differentially expressed genes (DEGs) related to membrane. The results of the phenotype tests and RNA-seq both focused on cell membrane damage, which showed that CA exerted antibacterial effect mainly by acting on cell membrane.

Fuzhuan brick tea (FBT) is a unique post-fermented tea product, naturally co-fermented by microorganisms, and has gained global popularity due to its potential health benefits for humans. Considerable efforts have been made toward elucidating the microbial diversity within FBT, but an understanding of the underlying FBT community interactions and functions remains poorly studied. Consequently, the microbial communities of two types of FBT, originating from Hunan and Shaanxi provinces, were investigated using comparative shotgun metagenomic sequencing and functional annotations. Metagenomic analysis indicated that two communities shared similar taxonomic and functional attributes. Two samples shared 486 genera, in which Pseudomonas contributed most to the abundant functions within the two samples. The carbohydrate active enzyme functions of the communities primarily comprised GH (32.92%), GT (26.8%), CEs (20.43%), and AAs (18.04%). Furthermore, the overall metabolic pathways encoded by the metagenomes were largely associated with carbohydrate and amino acid metabolism, with nine metabolic pathways that were differential between two groups including penicillin and cephalosporin biosynthesis. Significantly, a total of 35 potential probiotics were inferred, with Pseudomonas putida being the most abundant inferred probiotic (80%) within the FBT communities. This study provides new insights into FBT microbial communities on their potential functions and roles in FBT characteristics.

Alicyclobacillus acidoterrestris (A. acidoterrestris), a spore-forming bacterium, has become a main challenge and concern for the juices and acid beverage industry across the world due to its thermo-acidophilic characteristic. Thymoquinone (TQ) is one of the active components derived from Nigella sativa seeds. The objective of this study was to investigate antibacterial activity and associated molecular mechanism of TQ against A. acidoterrestris vegetative cells, and to evaluate effects of TQ on A. acidoterrestris spores and biofilms formed on polystyrene and stainless steel surfaces. Minimum inhibitory concentrations of TQ against five tested A. acidoterrestris strains ranged from 32 to 64 μg/mL. TQ could destroy bacterial cell morphology and membrane integrity in a concentration-dependent manner. Field-emission scanning electron microscopy observation showed that TQ caused abnormal morphology of spores and thus exerted a killing effect on spores. Moreover, TQ was effective in inactivating and removing A. acidoterrestris mature biofilms. These findings indicated that TQ is promising as a new alternative to control A. acidoterrestris and thereby reduce associated contamination and deterioration in the juice and acid beverage industry.

BackgroundDirect transdifferentiation of adult somatic cells into insulin-producing cells (IPCs) is a promising approach for cell-based therapies for type 1 diabetes mellitus. Liver cells are an ideal source for generating IPCs because they have regenerative ability and a developmental process similar to that of the pancreas. Pancreas versus liver fate is regulated by TALE homeoprotein (TGIF2) during development. Here, we wanted to investigate whether TGIF2 could enhance the efficiency of transdifferentiation of hepatocytes into IPCs induced by three pancreatic transcription factors (pTFs), i.e., Pdx1, NeuroD, and Mafa, which are crucial for pancreatic development in the embryo.MethodsThe in vitro transcribed (IVT) mRNAs of TGIF2 and the three pTFs were synthesized in vitro and sequentially supplemented in hepatocytes. On day 6, the expression of transcription factors was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and insulin expression was detected by immunofluorescence. Glucose-stimulated insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). The key genes controlling cell polarity and the Wnt/PCP signaling pathway were assayed by qRT-PCR, and the level of JNK protein phosphorylation, which regulates the Wnt/PCP signaling pathway, was detected by western blotting.ResultsIVT mRNAs could be efficiently transfected into hepatocytes. Quantitative real-time polymerase chain reaction results revealed that compared with ectopic expression of the three pTFs alone, ectopic expression of the three pTFs plus TGIF2 could strongly reduce hepatic gene expression and subsequently improve the induction of a set of pancreatic genes. Immunofluorescence analysis showed that TGIF2 expression could double the transdifferentiation yield; 30% of the cells were insulin positive if induced by TGIF2 plus the 3 pTFs, while only 15% of the cells were insulin positive if induced by the three pTFs alone. ELISA analysis confirmed that glucose-stimulated insulin secretion was less efficient after transfection with the three pTFs alone. The differentiated cells derived from the addition of TGIF2 mRNA could form islet-like clusters. By contrast, the cells differentiated with the three pTFs did not form clusters under the same conditions. Tgif2 induced transdifferentiation more efficiently by remodeling the expression of genes in the Wnt/PCP pathway. Overexpression of TGIF2 in hepatocytes could activate the expression of key genes controlling cell polarity and genes in the Wnt/PCP signaling pathway, increasing the level of JNK protein phosphorylation.ConclusionsOur study established a novel footprint-free protocol for efficient transdifferentiation of hepatocytes into IPCs using IVT mRNAs of TGIF2 and 3 pTFs, which paved the way toward a clinical application.

Probiotic strain Eurotium cristatum was isolated from Chinese Fuzhuan brick-tea and tested for its in vitro activity against aflatoxigenic Aspergillus flavus. Results indicated that E. cristatum can inhibit the radial growth of A. flavus. Furthermore, this inhibition might be caused by E. cristatum secondary metabolites. The ability of culture filtrate of strain E. cristatum against growth and aflatoxin B1 production by toxigenic A. flavus was evaluated in vitro. Meanwhile, the influence of filtrate on spore morphology of A. flavus was analyzed by scanning electron microscopy (SEM). Results demonstrated that both radial growth of A. flavus and aflatoxin B1 production were significantly weakened following increases in the E. cristatum culture filtrate concentration. In addition, SEM showed that the culture filtrate seriously damaged hyphae morphology. Gas chromatography mass spectrometry (GC/MS) analysis of the E. cristatum culture supernatant revealed the presence of multiple antifungal compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that the expression of aflatoxin biosynthesis-related genes (aflD, aflQ, and aflS) were down-regulated. Importantly, this latter occurrence resulted in a reduction of the AflS/AflR ratio. Interestingly, cell-free supernatants of E. cristatum facilitated the effective degradation of aflatoxin B1. In addition, two degradation products of aflatoxin B1 lacking the toxic and carcinogenic lactone ring were identified. A toxicity study on the HepG2 cells showed that the degradation compounds were less toxic when compared with AFB1.

The osmotolerant Zygosaccharomyces rouxii is known for its trait to survive in extreme high sugar environments. This ability determines its role in the fermentation process and leads to yeast spoilage in the food industry. However, our knowledge of the gene expression in response to high sugar stress remains limited. Here, we conducted RNA-sequencing (RNA-seq) under different sugar concentrations of the spoilage yeast, Z. rouxii, which exhibit extremely high tolerance to sugar stress. The obtained differentially expressed genes (DEGs) are significantly different to that of the Saccharomyces cerevisiae, which is sensitive to extreme high sugar stress. Most of the DEGs participated in the “glucan synthesis,” “transmembrane transport,” “ribosome,” etc. In this work, we also demonstrated that the gene ZYRO0B03476g (ZrKAR2) encoding Kar2p can significantly affect the growth of Z. rouxii under high sugar stress. In addition, we combined with a previous study on the genome sequence of Z. rouxii, indicating that several gene families contain significantly more gene copies in the Z. rouxii lineage, which involved in tolerance to sugar stress. Our results provide a gene insight for understanding the high sugar tolerance trait, which may impact food and biotechnological industries and improve the osmotolerance in other organisms.

As natural occurring antimicrobial substances, phenolic compounds have been used to inhibit various bacteria. Stenotrophomonas maltophilia 4–1, a strain isolated from food, exhibited spoilage potential in vitro with proteolysis and lipolysis at 25°C. The present study evaluated the antibacterial properties of 13 polyphenols on S. maltophilia 4–1, and selected 6 compounds (ferulic acid, p-coumaric acid, caffeic acid, chlorogenic acid, (−)-epigallocatechin, and phloretin) for binary combination treatments. The results revealed that antibacterial activities of polyphenols were structure-dependent, and cinnamic acid showed strong inhibitory effects, with a minimum inhibitory concentration (MIC) of 0.125 mg/mL. Importantly, we did not observe any obvious synergistic effects across all binary combinations. The antibacterial mechanism of cinnamic acid was related to membrane damage, caused by the loss of cell membrane integrity and alteration of cell morphology. These findings suggest that cinnamic acid is a promising candidate for the control of spoilage bacteria in food.

Alicyclobacillus acidoterrestris is a major putrefying bacterium that can cause pecuniary losses in the global juice industry. Current detection approaches are time-consuming and exhibit reduced specificity and sensitivity. In this study, an immunoproteomic approach was utilized to identify specific biomarkers from A. acidoterrestris for the development of new detection methods. Cell surface-associated proteins were extracted and separated by 2-D (two-dimensional) gel electrophoresis. Immunogenic proteins were detected by Western blot analysis using antisera against A. acidoterrestris. Twenty-two protein spots exhibiting immunogenicity were excised and eighteen of the associated spots were successfully identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS). These proteins were observed to be involved in energy and carbohydrate metabolism, transmembrane transport, response to oxidative stress, polypeptide biosynthesis, and molecule binding activity. This is the first report detailing the identification of cell surface-associated antigens of A. acidoterrestris. The identified immunogenic proteins could serve as potential targets for the development of novel detection methods.

Penicillium expansum, as a main postharvest pathogen of fruits, can secrete patulin (PAT), causing fruit decay and health problems. In this study, the antifungal test, SEM (scanning electron microscope) observation, transcriptional profile, PAT biosynthesis, and physiological characters of P. expansum exposed to cinnamaldehyde and citral combination (Cin/Cit) were evaluated. Cin/Cit could inhibit the mycelial growth and spore germination of P. expansum in a dose-dependent manner. Besides, Cin/Cit caused spores and mycelia wrinkled and depressed by SEM observation. Gene expression profiles of P. expansum were conducted by RNA sequencing (RNA-seq) in the presence or absence of Cin/Cit treatment. A total of 1713 differentially expressed genes (DEGs) were obtained, including 793 down-regulated and 920 up-regulated genes. Most of the DEGs participated in the biosynthesis of secondary metabolites, amino acid metabolism, and oxidation-reduction process, etc. Cin/Cit induced the dysfunction of the mitochondrial membrane, causing the potential influence on energy metabolism and reactive oxidative species production. The changes of superoxide dismutase (SOD) and catalase (CAT) activities combing with the increase of hydrogen peroxide content indicated the oxidative stress on P. expansum induced by Cin/Cit, which corresponded well with the transcriptional results. Moreover, both the RNA-seq data and the qRT-PCR showed the remarkable down-regulation of genes included in the PAT biosynthetic pathway under the Cin/Cit treatment. These findings provided more useful information about the antifungal mechanism of Cin/Cit against P. expansum at molecular and gene levels and suggested that Cin/Cit is a potential candidate to control P. expansum.