R-loop-dependent promoter-proximal termination ensures genome stability.

The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.

Characteristics and formation mechanism of key volatile compounds in sugar-smoked chicken.

In this paper, the volatile compounds and profile of sugar-smoked chicken thighs were studied, which were proceed by using dividual smoking materials such as sucrose, maltose, glucose, fructose and xylose. A total of 33 volatile compounds in sugar-smoked samples were identified and quantitated by gas chromatography/mass spectrometry (GC/MS). The furfural, 5-methylfurfural, 1-octene-3-ol, hexanal, heptanal and nonanal were the most important volatile compounds with odour activity values (OAVs) greater than 1, among which furfural and 5-methylfurfural contributed mainly to the smoky aroma. The sucrose, glucose and fructose pyrolysis all produced high yield of furans, such as furfural (FF) and 5-hydroxymethylfurfural (5-HMF) based on pyrolysis mass spectrometry and gas chromatography/mass spectrometry (Py-GC/MS). During the smoking process, then the 5-HMF pyrolyzed into 5-methylfurfural. The sucrose was the good carbohydrate to form furans with smoky aroma. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05071-y.

RP5-1120P11.3 promotes hepatocellular carcinoma development via the miR-196b-5p-WIPF2 axis.

Hepatocellular carcinoma (HCC) remains a huge threat to human health even though the diagnosis and treatment strategies have improved rapidly in the past few decades. Increasing evidence has illustrated the critical role noncoding RNA and their regulatory network play in the pathology of HCC. Here, we identified a novel long noncoding RNA, RP5-1120P11.3, that is ectopically expressed in HCC. Further characterization of RP5-1120P11.3 revealed that it promoted proliferation and invasion of HCC cells while inhibiting apoptosis. Importantly, our data revealed that miR-196b-5p interacted with and was regulated by RP5-1120P11.3 via a sponging mechanism. Inhibition of miR-196b-5p attenuated the phenotypes resulting from RP5-1120P11.3 inhibition. Moreover, our data showed that miR-196b-5p inhibited the expression of WIPF2 in HCC, illustrating a regulatory axis of RP5-1120P11.3-miR-196b-5p-WIPF2 that facilitated the progression of HCC. In addition, our data showed that RP5-1120P11.3 contributed to xenograft generation in vivo by regulating miR-196b-5p and WIPF2. These findings suggested that the RP5-1120P11.3-miR-196b-5p-WIPF2 axis is a potential target for treatment of HCC.

Long Non-coding RNA LINC01420 Contributes to Pancreatic Cancer Progression Through Targeting KRAS Proto-oncogene.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been increasingly uncovered to participate in multiple human cancers, including pancreatic cancer (PC). However, the underlying mechanisms of most of the lncRNAs have not been fully understood yet. AIMS: In this study, we probed the role and latent mechanism of LINC01420 in PC. METHODS: Several online tools were applied. Gene expression was evaluated by qRT-PCR or Western blot. Both in vitro and in vivo assays were conducted to probe LINC01420 function in PC. ChIP, RIP, and luciferase reporter assays were performed to determine relationships between genes. RESULTS: The bioinformatics analyses revealed LINC01420 was highly expressed in PC tissues. Besides, LINC01420 was pronouncedly upregulated in PC cell lines and its depletion controlled PC cell proliferation and EMT in vitro and hindered tumor growth in vivo. Importantly, KRAS was proved to mediate LINC01420-facilitated PC cell proliferation. Further, we explained that KRAS transcription was regulated by MYC, while LINC01420 enhanced the binding of MYC to KRAS promoter in the nucleus of PC cells. Intriguingly, LINC01420 boosted MYC expression in the cytoplasm of PC cells by sponging miR-494-3p. CONCLUSION: This study illustrated that LINC01420 accelerates PC progression through releasing miR-494-3p-silenced MYC in cytoplasm and upregulating MYC-activated KRAS in nucleus, unveiling LINC01420 as a latent therapeutic strategy for PC patients.

A circular RNA derived from COL6A3 functions as a ceRNA in gastric cancer development.

Gastric cancer (GC) poses a serious threat to human life, whereas its pathogenesis remains elusive. The present study aimed to investigate the potential pathogenic mechanism behind gastric cancer development. RT-PCR analysis using divergent primers, RNase R digestion assay, and mRNA stability assay were performed to characterize circCOL6A3 (ID: hsa_circ_0006401) in GC cell lines; Western blot was conducted to detect the expression of COL6A3. Cell counting kit-8 (CCK-8), EdU incorporation assay, and Transwell were run to evaluate GC cell proliferation and migration. Luciferase reporter assay was performed to validate the relationship between miR-3064-5p and COL6A3. Both circCOL6A3 and COL6A3 were highly expressed in GC cells, while miR-3064-5p was down-regulated. Depleted circCOL6A3 significantly decreased cell viability and mobility, and increased cell apoptosis. CircCOL6A3 regulated the expression of miR-3064-5p, and the effect of si-circCOL6A3 on cell biological behaviors was abolished by miR-3064-5p inhibitor. MicroRNA-3064-5p targets COL6A3 to regulate its expression. Taken together, the present study indicated that overexpressed circCOL6A3 promoted cell proliferation, migration and apoptosis of gastric cancer through rescission of miR-3064-5p-induced inhibitory effect on COL6A3. Our study will furnish theoretical grounds for future clinical diagnosis and treatment of GC patients.

Myosin Heavy Chain-Associated RNA Transcripts Promotes Gastric Cancer Progression Through the miR-4529-5p/ROCK2 Axis.

BACKGROUND AND AIM: Characterization of genetic aberrations provides novel strategies for diagnosis and treatment of gastric cancer. Accumulating evidence has shown the involvement of long non-coding RNA (lncRNA) in the pathology of gastric cancer, especially in proliferation and metastasis. The aim of this study was to delineate the role of myosin heavy chain-associated RNA transcripts (MHRT), a heart-specific lncRNA, in gastric cancer and to understand the correlation between MHRT, miR-4529-5p, and ROCK2. METHODS: To study expression level of MHRT, clinical gastric cancer samples, gastric cancer cell lines, adjacent normal tissues, and gastric epithelial cell lines were used. Additionally, apoptosis, proliferation, and invasion of gastric cancer cells were studied with or without downregulation of MHRT and miR-4529-5p. RESULTS: We identified that MHRT was ectopically expressed in gastric cancer tissues and cell lines. Interestingly, similar to the anti-apoptotic role of MHRT in cardiomyocytes, our data illustrated that MHRT inhibits apoptosis of gastric cancer cells. Moreover, we found that MHRT promotes proliferation and invasion of gastric cancer cells in vitro. Importantly, our data revealed that MHRT regulates the expression of miR-4529-5p via direct binding. Additionally, functional experiments illustrated that miR-4529-5p is particularly responsible for MHRT-mediated regulation of apoptosis. Besides, ROCK2 was identified as a downstream target of miR-4529-5p. Additionally, upregulated MHRT promotes the expression of ROCK2 by inhibiting miR-4529-5p. CONCLUSION: Our data illustrated a MHRT/miR-4529-5p/ROCK2 regulatory axis that contributes to the tumorigenesis of gastric cancer and provided potential therapeutic targets for precise gastric cancer treatment.

Barriers for Deriving Transgene-Free Pig iPS Cells with Episomal Vectors.

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets.

PhI(OCOCF3)2-mediated intramolecular oxidative N-N bond formation: metal-free synthesis of 1,2,4-triazolo[1,5-a]pyridines.

The biologically important 1,2,4-triazolo[1,5-a]pyridines were readily synthesized from N-(pyridin-2-yl)benzimidamides via phenyliodine bis(trifluoroacetate)-mediated intramolecular annulation. This novel strategy allows for the convenient construction of a 1,2,4-triazolo[1,5-a]pyridine skeleton through direct metal-free oxidative N-N bond formation, featuring a short reaction time and high reaction yields.

Flavor profile analysis of grilled lamb seasoned with classic salt, chili pepper, and cumin (Cuminum cyminum) through HS-SPME-GC-MS, HS-GC-IMS, E-nose techniques, and sensory evaluation on Sonit sheep.

In this study, the volatile flavor profiles of grilled lamb seasoned with salt, chili pepper, and cumin were analyzed employing HS-SPME-GC-MS, HS-GC-IMS, E-nose, and sensory evaluation techniques. The E-nose was found effective in differentiating the samples seasoned variously. A total of 67 volatile compounds were identified by HS-SPME-GC-MS, and 59 by HS-GC-IMS. The PCA demonstrated a correlation between the seasonings and the volatile compounds, with five principal components accounting for 99.54% of the total variance. 1-octen-3-ol, 3-furanmethanol, acetic acid, and heptanal were introduced by salt; compounds like propyl acetate were correlated with chili pepper; a broader range, including ethyl 3-methylbutanoate and high concentrations of alpha-pinene, was associated with cumin. Samples seasoned with all three ingredients showed similarities to those associated with cumin, alongside unique compounds such as gamma-octalactone and alpha-pinene. Sensory evaluations by consumers indicated that the combination of these seasonings significantly enhanced the overall acceptability of the grilled lamb. PRACTICAL APPLICATION: Utilizing modern analytical techniques, this study has successfully revealed the distinct impacts of seasonings-salt, chili pepper, and cumin-on the flavor profile of grilled lamb. By providing experimental data on how each seasonings influence the flavor profile of grilled lamb prepared with Sonit sheep. The research offers theoretical foundation for the development of grilled lamb products. By conducting a thorough comparison between GC-MS and GC-IMS, this study has expanded the understanding of the distinct characteristics of these two technologies. It has also provided a clearer analysis of some flavor compounds dimers produced in GC-IMS system.

Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model.

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

TSA Activates Pluripotency Factors in Porcine Recloned Embryos.

Animal cloning is of great importance to the production of transgenic and genome-edited livestock. Especially for multiple gene-editing operations, recloning is one of the most feasible methods for livestock. In addition, a multiple-round cloning method is practically necessary for animal molecular breeding. However, cloning efficiency remains extremely low, especially for serial cloning, which seriously impedes the development of livestock breeding based on genome editing technology. The incomplete reprogramming and failure in oocyte activation of some pluripotent factors were deemed to be the main reason for the low efficiency of animal recloning. Here, to overcome this issue, which occurred frequently in the process of animal recloning, we established a reporter system in which fluorescent proteins were driven by pig OCT4 or SOX2 promoter to monitor the reprogramming process in cloned and recloned pig embryos. We studied the effect of different histone deacetylase (HDAC) inhibitors on incomplete reprogramming. Our results showed that Trichostatin A (TSA) could activate pluripotent factors and significantly enhance the development competence of recloned pig embryos, while the other two inhibitors, valproic acid (VPA) and Scriptaid, had little effect on that. Furthermore, we found no difference in OCT4 mRNA abundance between TSA-treated and untreated embryos. These findings suggest that TSA remarkably improves the reprogramming state of pig recloned embryos by restoring the expression of incompletely activated pluripotent genes OCT4 and SOX2.

7-Methoxyisoflavone ameliorates atopic dermatitis symptoms by regulating multiple signaling pathways and reducing chemokine production.

7-Met, a derivative of soybean isoflavone, is a natural flavonoid compound that has been reported to have multiple signaling pathways regulation effects. This study investigated the therapeutic effects of 7-Met on mice with atopic dermatitis induced by fluorescein isothiocyanate (FITC), or oxazolone (OXZ). 7-Met ameliorated FITC or OXZ-induced atopic dermatitis symptoms by decreasing ear thickness, spleen index, mast cell activation, neutrophil infiltration and serum IgE levels in female BALB/c mice. In FITC-induced atopic dermatitis mice, 7-Met reduced Th1 cytokines production and regulated Th1/Th2 balance by downregulating the secretion of thymic stromal lymphopoietin (TSLP) via inactivation of the NF-κB pathway. In OXZ-induced atopic dermatitis, 7-Met functioned through the reduction of Th17 cytokine production. Our study showed that 7-Methoxyisoflavone alleviated atopic dermatitis by regulating multiple signaling pathways and downregulating chemokine production.

Magnoflorine inhibits human gastric cancer progression by inducing autophagy, apoptosis and cell cycle arrest by JNK activation regulated by ROS.

The antitumor effect of magnoflorine (Mag), an alkaloid isolated from Coptidis Rhizoma, in gastric cancer (GC) cells has not been reported. In the study, Mag suppressed the proliferation of GC cells, but showed no influence on normal gastric cells. Mechanistically, Mag induced autophagy in GC cells, as evidenced by the up-regulated expression of LC3B-II and increased autophagosome formation. Furthermore, we found that Mag-triggered autophagic cell death was regulated by reactive oxygen species (ROS)-induced suppression of serine/threonine-protein kinases (AKT) signaling. What's more, Mag treatment led to apoptosis in GC cells through enhancing cleaved Caspase-3 and PARP expressions. In addition, up-regulated expression of p27 and p21, as well as down-regulated expression of Cyclin-A and Cyclin-B1 was detected in Mag-treated GC cells, contributing to the S/G2 cell cycle arrest. Importantly, Mag incubation resulted in a significant increase in jun N-terminal kinase (JNK) phosphorylation but not p38 and ERK1/2, which was involved in the modulation of apoptosis and S/G2 phase arrest. Moreover, ROS production was highly induced by Mag treatment, and Mag-exhibited these functions was largely dependent on the generation of ROS in GC cells. Consistently, the GC cell xenograft mouse model confirmed the anti-tumor role of Mag in vivo. Collectively, these results indicated that Mag showed anti-GC effects, which could be a potential therapeutic target for GC treatment.

Comparative analysis of characteristic volatile compounds in Chinese traditional smoked chicken (specialty poultry products) from different regions by headspace-gas chromatography-ion mobility spectrometry.

This article presents investigation of the flavor profile on 5 different regional Chinese smoked chicken samples using gas chromatography-ion mobility spectrometry analysis methods. Five batches of samples were obtained over the course of 6 mo. A total of 34 flavor substances were identified in the 5 smoked chicken samples, including 10 aldehydes, 7 alcohols, 4 ketones, 2 hydrocarbons, 3 heterocyclic compounds, 4 esters, 2 ethers, and 2 phenolic compounds. The whole spectral fingerprint visually displayed flavor differences and relations in 5 types of smoked chicken with local characteristics. Moreover, the orthogonal projections to latent structures discriminant analysis model revealed that these samples could be separately classified into 5 groups. Multivariate statistical analysis showed that 20 chemicals with higher Variable Importance for the Projection values were the key contributors to the differences of flavor in these 5 kinds of smoked chicken. N-nonanal, heptanal, n-nonanal, heptanal, furfurol, and hexanal were the main common flavor compounds in the 5 types of Chinese smoked chicken, whereas linalool, alpha-terpineol, 1,8-cineole, and anethole were the main characteristic flavor compounds of Goubangzi chicken (No. 1); gamma-butyrolactone, 2-acetylfuran, 2-methoxyphenol, 2-acetylpyrrole, and limonene were determined as the key flavor compounds of Liaocheng chicken (No. 2); the concentration of octanal and n-nonanal was higher in Tangqiao chicken (No. 3); butyl acetate was the key contributor to the flavor compounds of Jinshan chicken (No. 4). 2-Heptanone and 2-pentylfuran had a high correlation with Zhuozishan chicken (No. 5). The different raw materials and ingredients used, especially the method of preparation and cultural differences, in different regions of the country in China were the main reasons leading to the similarities and differences of volatile compounds in the 5 kinds of Chinese traditional smoked chicken.

Chicoric acid (CA) induces autophagy in gastric cancer through promoting endoplasmic reticulum (ER) stress regulated by AMPK.

Gastric cancer is one of the most common cancers leading to tumor-related deaths worldwide. Chicoric acid (CA) exhibits a variety of protective effects in different diseases. However, its role in regulating tumor progression has not been reported. Autophagy, as a conserved catabolic process, sustains cellular homoeostasis responding to stress to modulate cell fate. In the study, the effects of CA on gastric cancer were investigated. The results indicated that CA treatment markedly reduced the cell viability and induced apoptosis in gastric cancer cells, and prevented tumor growth in an established xenograft gastric cancer model. Furthermore, CA exposure significantly induced autophagy both in gastric cancer cells and tumor samples, as evidenced by the up-regulated expression of LC3II. Moreover, phosphorylated AMP-activated protein kinase (AMPK) and p70S6 kinase (p70s6k) expression were obviously promoted by CA in vitro and in vivo. Importantly, blocking AMPK activation abrogated CA-induced expression of LC3II in gastric cancer cells. In addition, endoplasmic reticulum (ER) stress in tumor samples or cells was markedly induced by CA treatment through promoting the expression of associated signals such as Parkin, protein kinase RNA-like ER kinase (PERK), activating transcription factors 4 (ATF4) and ATF6. Importantly, these effects were abolished by the inhibition of AMPK signaling. Collectively, our findings indicated that CA prevents human gastric cancer progression by inducing autophagy partly through the activation of AMPK, and represents an effective therapeutic strategy against gastric cancer development.

Dysfunction of splenic macrophages in cirrhotic patients with hypersplenism and HBV infection.

BACKGROUND: Hepatitis B virus (HBV) reinfection is a difficult problem to manage after liver transplantation in patients with cirrhosis. This study was designed to investigate the activation type of splenic macrophages in cirrhotic patients with hypersplenism and HBV infection to assess the immune function of splenic macrophages. METHODS: Fourteen cirrhotic patients with hypersplenism and HBV infection and 6 controls were enrolled in the study. Serum lipopolysaccharide (LPS) was detected with a limulus assay. The differential expression of cytokines by splenic tissue and splenic macrophages between the cirrhosis and control groups was compared with cytokine arrays. Furthermore, splenic macrophages were cultured and stimulated with LPS, after which tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 levels in the supernatant were determined. RESULTS: In cirrhotic patients, serum LPS levels increased significantly. Interferon-gamma, TNF-beta, and transforming growth factor-beta upregulated, whereas IL-4 and IL-13 levels did not change in splenic tissue. TNF-alpha upregulated significantly, whereas IL-4 and IL-5 levels had no significant changes in splenic macrophages. The IL-12 levels in culture media of splenic macrophages from cirrhotic patients were significantly lower than in controls after LPS stimulation. CONCLUSION: Splenic macrophages may be activated via incomplete M1 activation in cirrhotic patients with hypersplenism and HBV infection, and the immune function of splenic macrophages is impaired.

ß Cell Replacement after Gene Editing of a Neonatal Diabetes-Causing Mutation at the Insulin Locus.

Permanent neonatal diabetes mellitus (PNDM) can be caused by insulin mutations. We generated induced pluripotent stem cells from fibroblasts of a patient with PNDM and undetectable insulin at birth due to a homozygous mutation in the translation start site of the insulin gene. Differentiation of mutant cells resulted in insulin-negative endocrine stem cells expressing MAFA, NKX6.1, and chromogranin A. Correction of the mutation in stem cells and differentiation to pancreatic endocrine cells restored insulin production and insulin secretion to levels comparable to those of wild-type cells. Grafting of corrected cells into mice, followed by ablating mouse ß cells using streptozotocin, resulted in normal glucose homeostasis, including at night, and the stem cell-derived grafts adapted insulin secretion to metabolic changes. Our study provides proof of principle for the generation of genetically corrected cells autologous to a patient with non-autoimmune insulin-dependent diabetes. These cases should be readily amenable to autologous cell therapy.

Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA.

The whey protein ß-lactoglobulin (BLG) is a major milk allergen which is absent in human milk. Here, we for the first time generated DNA-free BLG bi-allelic knockout cow by zinc-finger nuclease (ZFNs) mRNA and produced BLG-free milk. According to the allergenicity evaluation of BLG-free milk, we found it can trigger lower allergic reaction of Balb/c mice including the rectal temperature drop and the allergen-specific immunoglobulin IgE production; BLG free-milk was easily digested by pepsin at 2 min, while BLG in control milk was still not completely digested after 60 min, and the binding of IgE from cow's milk allergy (CMA) patients to BLG free-milk was significantly lower than that to the control milk. Meanwhile, the genome sequencing revealed that our animal is free of off-target events. Importantly, editing animal genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to foods produced by genome edited animals. Finally, the ZFNs-mediated targeting in cow could be transmitted through the germline by breeding. These findings will open up unlimited possibilities of modifying milk composition to make it more suitable for human health and also improve the functional properties of milk.