Ectosomal PKM2 Promotes HCC by Inducing Macrophage Differentiation and Remodeling the Tumor Microenvironment.

Tumor-derived extracellular vesicles are important mediators of cell-to-cell communication during tumorigenesis. Here, we demonstrated that hepatocellular carcinoma (HCC)-derived ectosomes remodel the tumor microenvironment to facilitate HCC progression in an ectosomal PKM2-dependent manner. HCC-derived ectosomal PKM2 induced not only metabolic reprogramming in monocytes but also STAT3 phosphorylation in the nucleus to upregulate differentiation-associated transcription factors, leading to monocyte-to-macrophage differentiation and tumor microenvironment remodeling. In HCC cells, sumoylation of PKM2 induced its plasma membrane targeting and subsequent ectosomal excretion via interactions with ARRDC1. The PKM2-ARRDC1 association in HCC was reinforced by macrophage-secreted cytokines/chemokines in a CCL1-CCR8 axis-dependent manner, further facilitating PKM2 excretion from HCC cells to form a feedforward regulatory loop for tumorigenesis. In the clinic, ectosomal PKM2 was clearly detected in the plasma of HCC patients. This study highlights a mechanism by which ectosomal PKM2 remodels the tumor microenvironment and reveals ectosomal PKM2 as a potential diagnostic marker for HCC.

Therapeutic Inhibition of LincRNA-p21 Protects Against Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. METHODS: We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. RESULTS: lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1. lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21. CONCLUSIONS: These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.

Electrochemical Oxidation Enables Radical Dearomative Spiroannulation to 2H-Spiro[benzofuran-3,9'-fluoren]-2-one.

Developing pragmatic strategies for accessing functional benzofuran-2-ones from 3-([1,1'-biphenyl]-2-yl)benzofuran remains an enduring challenge. Herein, we have achieved a highly discriminating electrochemical oxidative dearomative spiroannulation of 3-([1,1'-biphenyl]-2-yl)benzofuran, culminating in the synthesis of 2H-spiro[benzofuran-3,9'-fluoren]-2-one derivatives. By harnessing the electrophilic intermediates of benzofuryl radical cations supported by DFT calculations, we attain exceptional regioselectivity while eliminating the need for stoichiometric oxidants. Mechanistic investigations reveal a sequence of events involving the benzofuran radical cation, encompassing the capture of H2O, nucleophilic arene attack, and subsequent deprotonation, ultimately yielding the final benzofuran-2-ones.

CFHR1 involvement in bile duct carcinoma: Insights from a data mining study.

The aim of this study is to investigate the role of CFHR1 in bile duct carcinoma (BDC) and its mechanism of action, and we hope that our analysis and research will contribute to a better understanding of cholangiocarcinoma (BDC) disease genesis, progression and the development of new therapeutic strategies. The prognostic receiver operating characteristic curve of CFHR1 was generated using survival ROC. The ROC curve for CFHR1 showed that there is a correlation between CFHR1 expression and clinicopathological parameters and has an impact on poor prognosis. STRING was used to predict the protein-protein interaction network of the identified genes, and the Microenvironment Cell Populations counter algorithm was used to analyze immune cell infiltration within the BDC. The combined analysis showed that CFHR1 was found to be upregulated in BDC tissues, along with a total of 20 related differentially expressed genes (DEGs) (8 downregulated and 12 upregulated genes). Also, the results showed that the expression of CFHR1 is correlated with immune cell infiltration in tumor and immune cell markers in BDC (P < 0.05). In addition, we have verified experimentally the biological function of CFHR1. These findings suggest that CFHR1 may be a prognostic marker and a potential therapeutic target for BDC. Information regarding the detailed roles of CFHR1 in BDC could be valuable for improving the diagnosis and treatment of this rare cancer.

Neohesperidin Alleviates the Neuropathic Pain Behavior of Rats by Downregulating the P2X4 Receptor.

Neuropathic pain (NP) is a type of chronic pain affecting 6-8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1ß. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.

Nrf2 and its dependent autophagy activation cooperatively counteract ferroptosis to alleviate acute liver injury.

Ferroptosis has been implicated in the pathophysiological progression of a variety of diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular antioxidant response and can counteract ferroptosis by inducing autophagy and targeting genes involved in iron metabolism and glutathione (GSH) synthesis/metabolism. This study investigated how Nrf2 and autophagy interact to prevent ferroptosis in acute liver injury under sulforaphane (SFN) intervention. The results showed that SFN could activate Nrf2 signaling pathway and its downstream target genes, promote cell autophagy, and then combat ferroptosis to alleviate liver injury. After inhibiting Nrf2, the autophagy activated by SFN almost disappeared, and the anti-ferroptosis effect was greatly weakened. After inhibiting autophagy, SFN can still activate Nrf2 and its downstream target gene, but solute carrier family 7 member 11 (SLC7A11) membrane transfer and its cystine transport ability are significantly weakened, thus ultimately attenuating the anti-ferroptosis effect of SFN. Further studies showed that Nrf2-dependent autophagy activation disrupted SLC7A11 binding to S93-phosphorylated coiled-coil myosin-like BCL2-interacting protein (BECN1) and increased SLC7A11 membrane transfer to combat ferroptosis. In conclusion, Nrf2-dependent autophagy activation is essential for promoting SLC7A11 membrane localization to inhibit ferroptosis. Activation of Nrf2 not only upregulates the expression of SLC7A11, glutathione peroxidase 4 (GPX-4) and autophagy-related proteins, but also destroys the binding of SLC7A11 and BECN1 by inducing autophagy, thereby promoting SLC7A11 membrane transfer and GSH synthesis, and finally suppressing ferroptosis. However, inhibition of autophagy had no significant effect on the expression of Nrf2 and downstream genes during SFN anti-liver injury intervention.

Cardiac CIP protein regulates dystrophic cardiomyopathy.

Heart failure is a leading cause of fatality in Duchenne muscular dystrophy (DMD) patients. Previously, we discovered that cardiac and skeletal-muscle-enriched CIP proteins play important roles in cardiac function. Here, we report that CIP, a striated muscle-specific protein, participates in the regulation of dystrophic cardiomyopathy. Using a mouse model of human DMD, we found that deletion of CIP leads to dilated cardiomyopathy and heart failure in young, non-syndromic mdx mice. Conversely, transgenic overexpression of CIP reduces pathological dystrophic cardiomyopathy in old, syndromic mdx mice. Genome-wide transcriptome analyses reveal that molecular pathways involving fibrogenesis and oxidative stress are affected in CIP-mediated dystrophic cardiomyopathy. Mechanistically, we found that CIP interacts with dystrophin and calcineurin (CnA) to suppress the CnA-Nuclear Factor of Activated T cells (NFAT) pathway, which results in decreased expression of Nox4, a key component of the oxidative stress pathway. Overexpression of Nox4 accelerates the development of dystrophic cardiomyopathy in mdx mice. Our study indicates CIP is a modifier of dystrophic cardiomyopathy and a potential therapeutic target for this devastating disease.

Single-use flexible bronchoscopes vs traditional reusable flexible bronchoscopes: a prospective controlled study.

BACKGROUND: Single-use flexible bronchoscopes(SFB) eliminate the risk of bronchoscopy-related infection compared with traditional reusable flexible bronchoscopes(RFB). At present, there is no comparative study between SFB and RFB in the aspects of biopsy and interventional therapy. This study aims to explore whether SFB can perform complex bronchoscopic procedures such as transbronchial biopsies just like RFB. METHODS: We conducted a prospective controlled study. A total of 45 patients who required bronchoscopic biopsy in our hospital from June 2022 to December 2022 were enrolled. The patients were divided into the SFB group and the RFB group, and routine bronchoscopy, bronchoalveolar lavage, and biopsy were performed respectively. Data on the time of routine bronchoscopy, the recovery rate of bronchoalveolar lavage fluid(BALF), biopsy time, and bleeding volume were collected. Then we used the two-sample t-test and the χ2 test to assess the performance differences between SFB and RFB. We also designed a questionnaire to compare the performance between SFB and RFB by different bronchoscope operators. RESULTS: The routine examination time of SFB and RFB was 3.40 ± 0.50 min and 3.55 ± 0.42 min, respectively. There was no significant difference between the two groups (P = 0.308). The recovery rate of BALF was (46.56 ± 8.22) % in the SFB group and (47.00 ± 8.07) in the RFB group, without a significant difference between the two groups(P = 0.863). The biopsy time was similar(4.67 ± 0.51 min VS 4.57 ± 0.45 min) in both groups, with no significant difference(P = 0.512). The positive biopsy rate was 100% in both groups, with no significant difference. Overall, the bronchoscope operators were generally satisfied with SFB. CONCLUSION: SFBs are non-inferior to RFBs in routine bronchoscopy, bronchoalveolar lavage, and biopsy. It is suggested that SFBs have a wider clinical application.

Regulation of myonuclear positioning and muscle function by the skeletal muscle-specific CIP protein.

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy in mdx mice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.

Resveratrol reverses TGF-ß1-mediated invasion and metastasis of breast cancer cells via the SIRT3/AMPK/autophagy signal axis.

Resveratrol (Resv) has antitumorigenic and antimetastatic activities; however, the molecular mechanisms underlying the inhibitory effects of Resv on the invasion and metastasis of breast cancer cells are still a subject of debate. In our study, we demonstrated that Resv inhibited tumor cell proliferation and tumor growth. It also suppressed invasion and pulmonary metastasis of breast cancer by reversing the transforming growth factor beta 1 (TGF-ß1)-mediated EMT process. Meanwhile, the anticarcinogenic effects of Resv were abolished by the autophagy blocker 3-methyladenine (3-MA) or Beclin 1 small interfering RNA. Moreover, Resv upregulated autophagy-related genes and protein levels and induced the formation of autophagosomes in 4T1 breast cancer cells and xenograft mice, suggesting that autophagy was involved in the anticarcinogenic activities of Resv in both models. In addition, Resv-induced autophagy by increasing the expression of SIRT3 and phosphorylated AMPK. SIRT3 knockdown reduced AMPK phosphorylation and autophagy-related proteins levels, and suppressed the anticancer effects of Resv, demonstrating that the inhibitory effects of Resv on tumor progression were mediated via the SIRT3/AMPK/autophagy pathway. Taken together, our study provided novel insight into the anticancer effects of Resv and revealed that targeting the SIRT3/AMPK/autophagy pathway can serve as a new therapeutic target against breast cancer.

Comparative transcriptomic analysis provides insights into the molecular basis underlying pre-harvest sprouting in rice.

BACKGROUND: Pre-harvest sprouting (PHS) is one of the most serious rice production constraints in areas where prolonged rainfall occurs during harvest. However, the molecular mechanisms of transcriptional regulation underlying PHS remain largely unknown. RESULTS: In the current study, comparative transcriptome analyses were performed to characterize the similarities and differences between two rice varieties: PHS-sensitive Jiuxiangzhan (JXZ) and PHS-resistant Meixiangxinzhan (MXXZ). The physiological experimental results indicated that PHS causes a significant decrease in starch content and, in contrast, a significant increase in soluble sugar content and amylase activity. The extent of change in these physiological parameters in the sensitive variety JXZ was greater than that in the resistant variety MXXZ. A total of 9,602 DEGs were obtained from the transcriptome sequencing data, and 5,581 and 4,021 DEGs were identified in JXZ and MXXZ under high humidity conditions, respectively. The KEGG pathway enrichment analysis indicated that many DEGs under high humidity treatment were mainly linked to plant hormone signal transduction, carbon metabolism, starch and sucrose metabolism, and phenylpropanoid biosynthesis. Furthermore, the number of upregulated genes involved in these pathways was much higher in JXZ than in MXXZ, while the number of downregulated genes was higher in MXXZ than in JXZ. These results suggest that the physiological and biochemical processes of these pathways are more active in the PHS-sensitive JXZ than in the PHS-resistant MXXZ. CONCLUSION: Based on these results, we inferred that PHS in rice results from altered phytohormone regulation, more active carbon metabolism and energy production, and enhanced phenylpropanoid biosynthesis. Our study provides a theoretical foundation for further elucidation of the complex regulatory mechanism of PHS in rice and the molecular breeding of PHS-resistant rice varieties.

Set7 Deletion Prevents Glucose Intolerance and Improves the Recovery of Cardiac Function After Ischemia and Reperfusion in Obese Female Mice.

BACKGROUND/AIMS: An obesogenic diet (high fat and sugar, low fiber) is associated with an increased risk for metabolic and cardiovascular disorders. Previous studies have demonstrated that epigenetic changes can modify gene transcription and protein function, playing a key role in the development of several diseases. The methyltransferase Set7 methylates histone and non-histone proteins, influencing diverse biological and pathological processes. However, the functional role of Set7 in obesity-associated metabolic and cardiovascular complications is unknown. METHODS: Wild type and Set7 knockout female mice were fed a normal diet or an obesogenic diet for 12 weeks. Body weight gain and glucose tolerance were measured. The 3T3-L1 cells were used to determine the role of Set7 in white adipogenic differentiation. Cardiac morphology and function were evaluated by histology and echocardiography. An ex vivo Langendorff perfusion system was used to model cardiac ischemia/reperfusion (I/R). RESULTS: Here, we report that Set7 protein levels were enhanced in the heart and perigonadal adipose tissue (PAT) of female mice fed an obesogenic diet. Significantly, loss of Set7 prevented obesogenic diet-induced glucose intolerance in female mice although it did not affect the obesogenic diet-induced increase in body weight gain and adiposity in these animals, nor did Set7 inhibition change white adipogenic differentiation in vitro. In addition, loss of Set7 prevented the compromised cardiac functional recovery following ischemia and reperfusion (I/R) injury in obesogenic diet-fed female mice; however, deletion of Set7 did not influence obesogenic diet-induced cardiac hypertrophy nor the hemodynamic and echocardiographic parameters. CONCLUSION: These data indicate that Set7 plays a key role in obesogenic diet-induced glucose intolerance and compromised myocardial functional recovery after I/R in obese female mice.

Intermolecular interactions, photocatalysis and THz-TDS interrelationships for lanthanide phosphine oxide complexes based on {PW12}.

A series of rare earth complexes containing (α-PW12O40)3- and PO ligand are synthesized by water bath in 70 °C, [Ln(OPPh3)4(H2O)3](PW12O40)·4CH3CN (Ln = La, Pr, Nd, Sm, Gd, Tb, Ho 1-7) (OPPh3 = Triphenylphosphine oxide, {PW12} = phosphotungstic acid). The precise structures are confirmed by X-ray single crystal diffraction and the result shows all complexes are isostructural. Complexes 1-7 are fully characterized by PXRD, FT-IR, TGA, UV diffuse reflectance spectra and terahertz time-domain spectroscopy (THz-TDS). Complex 3 exhibits the highest photocatalytic degradation efficiency for methylene blue (MB) in this series of complexes. The experimental results showed that the photodegradation efficiency can remain constant at the level of 95% after five consecutive cycles. The photocatalytic reaction kinetics and mechanism of complexes were investigated. Additionally, complexes also exhibit photocatalytic hydrogen evolution activity. THz-TDS was used to characterize the complexes and its raw materials, the characteristic peaks of OPPh3 (broad peak at 1.20 THz) and phosphotungstic acid (sharp peaks at 0.23, 0.32 THz) were obtained.

Facile high yield, excellent catalytic performance of polyoxometalate-based lanthanide phosphine oxide complexes: Syntheses, structures, photocatalysis and THz spectra.

Water pollution, which continuously threatens human health and the sustainable development of society, has become a major concern. Photocatalytic degradation is an effective strategy to remove organic dyes from wastewater. For this strategy, it is crucial to select the appropriate catalyst. Using triphenylphosphine oxide (OPPh3) as the ligand, phosphomolybdic acid as the anion template, three new lanthanide complexes [Ln(OPPh3)4(H2O)3](PMo12O40)∙4C2H5OH (1-3) (Ln = Sm, Gd, Tb) were synthesized. The raw materials for the reaction are cheap and readily available. The convenient synthesis method is environmentally friendly, with high yield (70%-80%). Complexes 1-3 are all seven-coordinated mononuclear structures centered on lanthanide ions, [PMo12O40]3- anions and solvent molecules are not coordinated with metal ions. These mononuclear structures eventually form complicated 3D supramolecular structures through hydrogen bonds, Mo-O … π or C-H … π weak interactions. Complexes 1-3 photocatalytic degradation of MB have high removal rates, as catalysts have enough stability to be reused, and can be used as excellent catalysts for the degradation of dye molecules in sewage. Among them, the removal rate of MB by photodegradation of complex 2 was highest (99.50%). In addition, the effects of different initial concentrations of MB solution and different types of organic dyes on the photocatalysis experiment were investigated. The photocatalytic reaction mechanism of complexes 1-3 was also studied. Due to the similar structures of complexes 1-3, they have almost the same THz absorption spectra with different absorption intensity, which may be attributed to the difference of the number of weak interactions. Therefore, terahertz spectroscopy can be used as a sensitive method to distinguish and determine small differences between lanthanide-organic complexes. This is the first time that this spectrum has been used to characterize lanthanide phosphine oxide complexes modified by [PMo12O40]3-.

Innate antiviral host defense attenuates TGF-ß function through IRF3-mediated suppression of Smad signaling.

TGF-ß signaling is essential in many processes, including immune surveillance, and its dysregulation controls various diseases, including cancer, fibrosis, and inflammation. Studying the innate host defense, which functions in most cell types, we found that RLR signaling represses TGF-ß responses. This regulation is mediated by activated IRF3, using a dual mechanism of IRF3-directed suppression. Activated IRF3 interacts with Smad3, thus inhibiting TGF-ß-induced Smad3 activation and, in the nucleus, disrupts functional Smad3 transcription complexes by competing with coregulators. Consequently, IRF3 activation by innate antiviral signaling represses TGF-ß-induced growth inhibition, gene regulation and epithelial-mesenchymal transition, and the generation of Treg effector lymphocytes from naive CD4(+) lymphocytes. Conversely, silencing IRF3 expression enhances epithelial-mesenchymal transition, TGF-ß-induced Treg cell differentiation upon virus infection, and Treg cell generation in vivo. We present a mechanism of regulation of TGF-ß signaling by the antiviral defense, with evidence for its role in immune tolerance and cancer cell behavior.

SOX9/MKLN1-AS Axis Induces Hepatocellular Carcinoma Proliferation and Epithelial-Mesenchymal Transition.

SOX9, as a transcript factor, has been confirmed to boost proliferation and epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC), but the underlying mechanism remains incompletely elucidated. A bioinformatics analysis web, Jaspar, manifested that SOX9 can transcriptionally regulate an lncRNA, MKLN1-AS. To determine the role of MKLN1-AS in HCC, this study measured MKLN1-AS expression in HCC and the paracancerous tissues and conducted a series of assays, including MTT, colony formation, and transwell assays, in vitro. EMT of HCC was evaluated by E-cadherin and vimentin protein levels. The regulatory effect of SOX9 on MKLN1-AS was determined using dual luciferase reporter and ChIP assays. Both MKLN1-AS and SOX9 were up-regulated in HCC tissues compared to paracancerous tissues. SOX9 promoted cell viability, proliferation, invasion, and EMT of HCCs, but these promoting effects of SOX9 were attenuated after the knockdown of MKLN1-AS. Overexpression of SOX9 increased MKLN1-AS in HCCs, whereas silencing SOX9 decreased MKLN1-AS expression. According to dual luciferase reporter and ChIP assays, SOX9 can bind to the promoter of MKLN1-AS gene to stimulate the expression. MKLN1-AS is transcriptionally regulated by SOX9 and mediates the effects of SOX9 on the proliferation and EMT of HCCs.

Food grade microbial synthesis of the butter aroma compound butanedione using engineered and non-engineered Lactococcus lactis.

The design-build-test-learn (DBTL) cycle has been implemented in metabolic engineering processes for optimizing the production of valuable compounds, including food ingredients. However, the use of recombinant microorganisms for producing food ingredients is associated with different challenges, e.g., in the EU, a content of more than 0.9% of such ingredients requires to be labeled. Therefore, we propose to expand the DBTL cycle and use the "learn" module to guide the development of non-engineered strains for clean label production. Here, we demonstrate how this approach can be used to generate engineered and natural cell factories able to produce the valuable food flavor compound - butanedione (diacetyl). Through comprehensive rerouting of the metabolism of Lactococcus lactis MG1363 and re-installment of the capacity to metabolize lactose and dairy protein, we managed to achieve a high titer of diacetyl (6.7 g/L) in pure dairy waste. Based on learnings from the engineering efforts, we successfully achieved the production of diacetyl without using recombinant DNA technology. We accomplish the latter by process optimization and by relying on high-throughput screening using a microfluidic system. Our results demonstrate the great potential that lies in combining metabolic engineering and natural approaches for achieving efficient production of food ingredients.

Adaptive Laboratory Evolution as a Means To Generate Lactococcus lactis Strains with Improved Thermotolerance and Ability To Autolyze.

Lactococcus lactis subsp. lactis (referred to here as L. lactis) is a model lactic acid bacterium and one of the main constituents of the mesophilic cheese starter used for producing soft or semihard cheeses. Most dairy L. lactis strains grow optimally at around 30°C and are not particularly well adapted to the elevated temperatures (37 to 39°C) to which they are often exposed during cheese production. To overcome this challenge, we used adaptive laboratory evolution (ALE) in milk, using a setup where the temperature was gradually increased over time, and isolated two evolved strains (RD01 and RD07) better able to tolerate high growth temperatures. One of these, strain RD07, was isolated after 1.5 years of evolution (400 generations) and efficiently acidified milk at 41°C, which has not been reported for industrial L. lactis strains until now. Moreover, RD07 appeared to autolyze 2 to 3 times faster than its parent strain, which is another highly desired property of dairy lactococci and rarely observed in the L. lactis subspecies used in this study. Model cheese trials indicated that RD07 could potentially accelerate cheese ripening. Transcriptomics analysis revealed the potential underlying causes responsible for the enhanced growth at high temperatures for the mutants. These included downregulation of the pleiotropic transcription factor CodY and overexpression of genes, which most likely lowered the guanidine nucleotide pool. Cheese trials at ARLA Foods using RD01 blended with the commercial Flora Danica starter culture, including a 39.5°C cooking step, revealed better acidification and flavor formation than the pure starter culture. IMPORTANCE In commercial mesophilic starter cultures, L. lactis is generally more thermotolerant than Lactococcus cremoris, whereas L. cremoris is more prone to autolysis, which is the key to flavor and aroma formation. In this study, we found that adaptation to higher thermotolerance can improve autolysis. Using whole-genome sequencing and RNA sequencing, we attempt to determine the underlying reason for the observed behavior. In terms of dairy applications, there are obvious advantages associated with using L. lactis strains with high thermotolerance, as these are less affected by curd cooking, which generally hampers the performance of the mesophilic starter. Cheese ripening, the costliest part of cheese manufacturing, can be reduced using autolytic strains. Thus, the solution presented here could simplify starter cultures, make the cheese manufacturing process more efficient, and enable novel types of harder cheese variants.

Long non-coding RNA muskelin 1 antisense RNA (MKLN1-AS) is a potential diagnostic and prognostic biomarker and therapeutic target for hepatocellular carcinoma.

BACKGROUNDS/AIMS: Hepatocellular carcinoma is recognized as the most common subtype of hepatic cancer. Muskelin 1 antisense RNA (MKLN1-AS) shows prognostic value in hepatitis B virus-hepatocellular carcinoma. The aim of this study is to investigate the detailed biological role of MKLN1-AS and Yes-associated transcriptional regulator 1 (YAP1)-related mechanisms. METHODS: Based on online databases (GEPIA, TCGA, and GEO), the expression of MKLN1-AS and YAP1 in patients with hepatocellular carcinoma was analyzed. The IntaRNA algorithm was used to predict complementary sites between MKLN1-AS and YAP1 mRNA. Hepatocellular carcinoma tumor tissues and cells were collected for the quantification of MKLN1-AS and YAP1. FISH was performed to explore the location of MKLN1-AS in cells. The effects of MKLN1-AS and YAP1 on proliferation, migration and invasionof hepatocellular carcinoma were determined in vitro and in vivo. Actinomycin D and RNA immunoprecipitation were resorted to confirm the regulatory role of MKLN1-AS in YAP1 expression. RESULTS: The up-regulation of MKLN1-AS contributed to the poor prognosis of patients with hepatocellular carcinoma. MKLN1-AS and YAP1 were overexpressed in hepatocellular carcinoma tissues and cells. MKLN1-AS positively modulated YAP1 expression through targeting and stabilizing YAP1 mRNA.MKLN1-AS was predominantly located in the cytoplasm of the cells. MKLN1-AS intensified proliferation, migration and invasion of hepatocellular carcinoma cells via YAP1. MKLN1-AS also caused hepatocarcinogenesis through inducing YAP1 expression in vivo. CONCLUSIONS: MKLN1-AS overexpression enhances the stability of YAP1 mRNA, which is necessary for the oncogenic activity of MKLN1-AS. MKLN1-AS can be utilized in the diagnosis and prognosis of hepatocellular carcinoma as an upstream factor of YAP1.

Electrooxidative double C-H/C-H coupling of phenols with 3-phenylbenzothiophenes: facile access to benzothiophene derivatives.

An efficient electrooxidative double C-H/C-H coupling of phenols with 3-phenylbenzothiophene has been developed under external oxidant- and catalyst-free conditions. This strategy could enable the highly tunable access to benzothiophene derivatives and exhibited broad substrate generality under mild conditions. The reaction is likely to proceed via the cross-coupling of the p-methoxylphenol radical and the 3-phenylbenzothiophene radical cation.