Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation.

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-ß-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

Potential of animal-welfare compliant and sustainably sourced serum from pig slaughter blood.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

Fetal bovine serum contains biologically available ATP.

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Interactions Between Silver Nanoparticles and Culture Medium Biomolecules with Dose and Time Dependencies.

The interaction between silver nanoparticles (AgNPs) and molecules producing coronas plays a key role in cytotoxicity mechanisms. Once adsorbed coronas determine the destiny of nanomaterials in vivo, their effective deployment in the biomedical field requires a comprehensive understanding of the dynamic interactions of biomolecules with nanoparticles. In this work, we characterized 40 nm AgNPs in three different nutritional cell media at different molar concentrations and incubation times to study the binding mechanism of molecules on surface nanoparticles. In addition, their cytotoxic effects have been studied in three cell lineages used as tissue regeneration models: FN1, HUV-EC-C, RAW 264.7. According to the data, when biomolecules from DMEM medium were in contact with AgNPs, agglomeration and precipitation occurred. However, FBS medium proteins indicated the formation of coronas over the nanoparticles. Nonetheless, little adsorption of molecules around the nanoparticles was observed when compared to DMEM supplemented with 10% FBS. These findings indicate that when nanoparticles and bioproteins from supplemented media interact, inorganic salts from DMEM contribute to produce large bio-coronas, the size of which varies with the concentration and time. The static quenching mechanism was shown to be responsible for the fluorescence quenching of the bioprotein aggregates on the AgNPs surface. The calculated bioprotein-nanoparticle surface binding constants were on the order of 105 M-1 at 37 °C, with hydrophobic interactions driven by enthalpy and entropy playing a role, as confirmed by thermodynamic analysis. Cytotoxicity data showed a systematic degrowth in the viable cell population as the number of nanoparticles increased and the diameter of coronas decreased. Cytotoxic intervals associated with half decrease of cell population were established for AgNPs molar concentration of 75 µM for 24 h and 50 µM for 48 h. In summary, through the cytotoxicity mechanism of bio-coronas we are able to manipulate cells' expansion rates to promote specific processes, such inflammatory mechanisms, at different time instants.

The effect of psyllium on fasting blood sugar, HbA1c, HOMA IR, and insulin control: a GRADE-assessed systematic review and meta-analysis of randomized controlled trials.

There is equivocal evidence that psyllium can prevent or attenuate increases in fasting blood sugar. Therefore, this systematic review and meta-analysis sought to investigate the influence of psyllium on hemoglobin A1C (HbA1c), fasting blood sugar (FBS), insulin, and Homeostatic Model Assessment of Insulin Resistance (HOMA IR). We searched PubMed, ISI Web of Science (WOS), and Scopus for eligible publications, up to 15 July 2022, including randomized controlled trials (RCT) assessing the effect of psyllium on HbA1c, FBS, insulin, and HOMA IR levels in adults. Using a random effects model, we report the weighted mean differences (WMD) with 95% confidence intervals (CI). In this article, 19 RCT studies, consisting of 962 participants, were included. Psyllium significantly decreased FBS, HbA1c, and HOMA IR levels, but not insulin levels, as compared to placebo (FBS: WMD): -6.89; 95% CI: -10.62, -3.16; p < .001), HbA1c: (WMD: -0.75; 95% CI: -1.21, -0.29; p < .001), HOMA IR: (WMD: -1.17; 95% CI: -2.11, -0.23; p < .05), and insulin: (WMD: -2.08; 95% CI: -4.21, -0.035; p > .05)). Subgroup analyses illustrated differences in the effects of psyllium on FBS: dosages less than and more than 10 g/d showed significant differences (p value < 0.05). However, it was not significant in intervention durations less than 50 days (p value > 0.05). For HbA1c: psyllium consumption less than 10 g/d (p value > 0.05) was non-significant. For HOMA IR and insulin: no significant changes were noted with psyllium consumption less than vs. more than 10 g/d. In conclusion, we found that psyllium could significantly decrease FBS, HbA1c, and HOMA IR levels, but not insulin levels, as compared to placebo.

Loss of peptide:N-glycanase causes proteasome dysfunction mediated by a sugar-recognizing ubiquitin ligase.

Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de-N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2-like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum-associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCFFBS2 in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of "glycan-less" NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCFFBS2 accounts for the pathogenesis resulting from NGLY1 deficiency.

Effect of silymarin on lipid profile and glycemic control in patients with type 2 diabetes mellitus.

This randomized clinical trial was conducted to evaluate the effects of silymarin supplementation on glycemic indices and serum lipid profile in type 2 diabetes mellitus (T2DM) patients. In this open-label randomized clinical trial study, 48 patients with T2DM were eligible to participate for 12 weeks and were divided into two groups randomly: 24 subjects in the intervention (received three 140 mg silymarin capsules daily and diet plan) and 24 in control (received a diet plan). Fasting blood samples and anthropometric data were collected, and glycemic indices and lipid profiles were determined at baseline and at the end of the study. Out of 60 patients included in the clinical trial, 48 people completed the study. In comparing silymarin and control groups before and after the study, a significant reduction was observed in weight and body mass index. However, after adjustment, no significant difference was seen between the two groups. Furthermore, daily consumption of three capsules of 140 mg silymarin for 12 weeks did not show any significant difference on the level of fasting blood sugar (p = 0.789), HbA1c (p = 0.719), and lipid profile. The findings of the present study show that silymarin did not lead to changes in the level of glycemic index and lipid profile in patients with T2DM.

Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Evaluating the Impact of Intensifying Treatment from Human to Analogue Insulin on Glycaemic Control and Insulin Expenditure in Patients with Type 2 Diabetes: A Retrospective Cohort Study.

Background: Achieving good glycaemic control is essential to reducing the risk of diabetes complications. Insulin is the most effective therapy for achieving good glycaemic control; however, it is associated with a higher risk of hypoglycaemia, especially with human insulin. This study aimed to evaluate the efficacy of intensification from human to analogue insulin and its added cost. Methods: This retrospective study was conducted at the Hospital Universiti Sains Malaysia (HUSM). Patients with type 2 diabetes mellitus (T2DM) who underwent intensification for at least 3 months from human to analogue insulin were included in this study. The patients' medical records, haemoglobin A1c (Hba1c) and fasting blood sugar (FBS) were retrieved. The total cost pre- and post-intensification of insulin was obtained from the pharmacy database. Differences in HbA1c, FBS and total insulin cost pre- and post-intensification were analysed. Results: A total of 163 patients with T2DM who had intensification from human to analogue insulin were included in this study. HbA1c and FBS levels were significantly lower in analogue insulin. However, the differences were not clinically significant, as the mean reduction in HbA1c was less than 0.5%. Meanwhile, the total costs of analogue insulin for 3 months were higher. Conclusion: There were no clinically significant improvements in patients' HbA1c and FBS after the intensification of insulin, despite the extra costs spent. Hence, it is vital to choose the right group of patients to receive an insulin analogue to maximise its benefit but at the most optimal cost.

Health literacy, self-efficacy, self-care behaviors, and glycemic control among older adults with type 2 diabetes mellitus: a cross-sectional study in Thai communities.

BACKGROUND: Properly understanding the health information of people with type 2 diabetes mellitus (T2DM) is the basis for better risk factor management, which also positively affects their quality of life. The aim of this study was to investigate diabetes health literacy (HL), self-efficacy, and self-care behaviors associated with glycemic control among older adults with T2DM in northern Thai communities. METHODS: A cross-sectional study was conducted among 414 older adults over the age of 60 diagnosed with T2DM. The study was conducted in Phayao Province from January to May 2022. Simple random sampling of the patient list for the Java Health Center Information System program was used. Questionnaires were used to collect data on diabetes HL, self-efficacy, and self-care behaviors. Blood samples were tested for estimated glomerular filtration rate (eGFR) and glycemic controls, such as fasting blood sugar (FBS) and glycated hemoglobin (HbA1c). RESULTS: The mean age of participants was 67.1 years. FBS (mean ± SD = 108.5 ± 29.5 mg/dL) and HbA1c (mean ± SD = 6.6 ± 1.2%) levels were found to be abnormal in 50.5% (≥ 126 mg/dL) and 17.4% (≥ 6.5%) of the subjects, respectively. There was a strong correlation between HL and self-efficacy (r = 0.78), HL and self-care behaviors (r = 0.76), and self-efficacy and self-care behaviors (r = 0.84). The eGFR was significantly correlated with diabetes HL (r = 0.23), self-efficacy (r = 0.14), self-care behaviors (r = 0.16), and HbA1c (r = -0.16) scores. Linear regression after adjusting for sex, age, education, DM duration, smoking, and drinking alcohol showed that FBS level was inversely associated with diabetes HL (Beta = -0.21, R2 = 11.0%), self-efficacy (Beta = -0.43, R2 = 22.2%), and self-care behavior (Beta = -0.35, R2 = 17.8%), whereas HbA1C level was negatively associated with diabetes HL (Beta = -0.52, R2 = 23.8%), self-efficacy (Beta = -0.39, R2 = 19.1%), and self-care behavior (Beta = -0.42, R2 = 20.7%). CONCLUSION: Diabetes HL was related to self-efficacy and self-care behaviors in elderly T2DM patients and was shown to influence their health, including glycemic control. These findings suggest that implementing HL programs to build competence in self-efficacy expectations is important for contributing to improvements in diabetes preventive care behaviors and HbA1c control.

Protein-liposome interactions: the impact of surface charge and fluidisation effect on protein binding.

At the dawn of a new nanotechnological era in the pharmaceutical field, it is very important to examine and understand all the aspects that influence in vivo behaviour of nanoparticles. In this point of view, the interactions between serum proteins and liposomes with incorporated anionic, cationic, and/or PEGylated lipids were investigated to elucidate the role of surface charge and bilayer fluidity in protein corona's formation. 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) liposomes with the presence or absence of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt) (DOTAP), and/or 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DPPE-PEG 5000) lipids were prepared by the thin-film hydration method. The evaluation of their biophysical characteristics was enabled by differential scanning calorimetry and dynamic and electrophoretic light scattering. The physicochemical characteristics of mixed liposomes were compared before and after exposure to foetal bovine serum (FBS) and were correlated to calorimetric data. Our results indicate protein binding to all liposomal formulations. However, it is highlighted the importance of surface charge and fluidisation effect to the extent of protein adsorption. Additionally, considering the extensive use of cationic lipids for innovative delivery platforms, we deem PEGylation a key parameter, because even in a small proportion can reduce protein binding, and thus fast clearance and extreme toxicity without affecting positive charge. This study is a continuation of our previous work about protein-liposome interactions and fraction of stealthiness (Fs) parameter, and hopefully a design road map for drug and gene delivery.

Design and Evaluation of the Extended FBS Model Based Gaze-Control Power Wheelchair for Individuals Facing Manual Control Challenges.

This study addresses the challenges faced by individuals with upper limb disadvantages in operating power wheelchair joysticks by utilizing the extended Function-Behavior-Structure (FBS) model to identify design requirements for an alternative wheelchair control system. A gaze-controlled wheelchair system is proposed based on design requirements from the extended FBS model and prioritized using the MosCow method. This innovative system relies on the user's natural gaze and comprises three levels: perception, decision making, and execution. The perception layer senses and acquires information from the environment, including user eye movements and driving context. The decision-making layer processes this information to determine the user's intended direction, while the execution layer controls the wheelchair's movement accordingly. The system's effectiveness was validated through indoor field testing, with an average driving drift of less than 20 cm for participates. Additionally, the user experience scale revealed overall positive user experiences and perceptions of the system's usability, ease of use, and satisfaction.

Functional changes in long-term incubated rat precision-cut lung slices.

BACKGROUND: Respiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days. METHODS: Rat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation. RESULTS: Cultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin. CONCLUSIONS: Overall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.

Addressing Animal Welfare Issues in Fetal Blood Collection for Fetal Bovine Serum Production.

There is ethical debate over whether fetal calves suffer when their dam is slaughtered and fetal blood extracted by cardiac puncture for fetal bovine serum (FBS) production. Yet, the serum industry does not follow best practice, as recommended by the European Food Safety Authority (EFSA), to avoid fetal distress. We discuss the key elements of this debate, and recommend how the serum industry can alter its practices to improve animal welfare.

Case Studies Exemplifying the Transition to Animal Component-free Cell Culture.

Cell culture techniques are strongly connected with modern scientific laboratories and production facilities. Thus, choosing the most suitable medium for the cells involved is vital, not only directly to optimise cell viability but also indirectly to maximise the reliability of the experiments performed with the cells. Fetal bovine or calf serum (FBS or FCS, respectively) is the most commonly used cell culture medium supplement, providing various nutritional factors and macromolecules essential for cell growth. Yet, the use of FBS encompasses a number of disadvantages. Scientifically, one of the most severe disadvantages is the lot-to-lot variability of animal sera that hampers reproducibility. Therefore, transitioning from the use of these ill-defined, component-variable, inconsistent, xenogenic, ethically questionable and even potentially infectious media supplements, is key to achieving better data reproducibility and thus better science. To demonstrate that the transition to animal component-free cell culture is possible and achievable, we highlight three different scenarios and provide some case studies of each, namely: i) the adaptation of single cell lines to animal component-free culture conditions by the replacement of FBS and trypsin; ii) the adaptation of multicellular models to FBS-free conditions; and (iii) the replacement of FBS with human platelet lysate (hPL) for the generation of primary stem/stromal cell cultures for clinical purposes. By highlighting these examples, we aim to foster and support the global movement towards more consistent science and provide evidence that it is indeed possible to step out of the currently smouldering scientific reproducibility crisis.

Expression of the C-Terminal Domain of Phospholipase Cß3 Inhibits Signaling via Gαq-Coupled Receptors and Transient Receptor Potential Channels.

Transient receptor potential (TRP) channels are cation channels that play a regulatory role in pain and thermosensation, insulin secretion, and neurotransmission. It has been proposed that activation of TRP channels requires phosphatidylinositol 4,5-bisphosphate, the major substrate for phospholipase C (PLC). We investigated whether inhibition of PLCß has an impact on TRP channel signaling. A genetic approach was used to avoid off-target effects observed when using a pharmacological PLCß inhibitor. In this study, we show that expression of PLCß1ct and PLCß3ct, truncated forms of PLCß1 or PLCß3 that contain the C-terminal membrane binding domains, almost completely blocked the signal transduction of a Gαq-coupled designer receptor, including the phosphorylation of ERK1/2. In contrast, expression of the helix-turn-helix motif (Hα1-Hα2) of the proximal C-terminal domain of PLCß3 did not affect Gαq-coupled receptor signaling. PLCß3ct expression impaired signaling of the TRP channels TRPM3 and TRPM8, stimulated with either prognenolone sulfate or icilin. Thus, the C-terminal domain of PLCß3 interacts with plasma membrane targets, most likely phosphatidylinositol 4,5-bisphosphate, and in this way blocks the biological activation of TRPM3 and TRPM8, which require interaction with this phospholipid. PLCß thus regulates TRPM3 and TRPM8 channels by masking phosphatidylinositol 4,5-bisphosphate with its C-terminal domain.

Genistein promotes M1 macrophage apoptosis and reduces inflammatory response by disrupting miR-21/TIPE2 pathway.

Cardiovascular diseases are a major cause of mortality, and vascular injury, a common pathological basis of cardiovascular disease, is deeply correlated with macrophage apoptosis and inflammatory response. Genistein, a type of phytoestrogen, exerts cardiovascular protective activities, but the underlying mechanism has not been fully elucidated. In this study, RAW264.7 cells were treated with genistein, lipopolysaccharide (LPS), nuclear factor-kappa B (NF-κB) inhibitor, and/or protein kinase B (AKT) agonist to determine the role of genistein in apoptosis and inflammation in LPS-stimulated cells. Simultaneously, high fat diet-fed C57BL/6 mice were administered genistein to evaluate the function of genistein on LPS-induced cardiovascular injury mouse model. Here, we demonstrated that LPS obviously increased apoptosis resistance and inflammatory response of macrophages by promoting miR-21 expression, and miR-21 downregulated tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) expression by targeting the coding region. Genistein reduced miR-21 expression by inhibiting NF-κB, then blocked toll-like receptor 4 (TLR4) pathway and AKT phosphorylation dependent on TIPE2, resulting in inhibition of LPS. Our research suggests that miR-21/TIPE2 pathway is involved in M1 macrophage apoptosis and inflammatory response, and genistein inhibits the progression of LPS-induced cardiovascular injury at the epigenetic level via regulating the promoter region of Vmp1 by NF-κB.

The Effect of Berberine on Lipid Profile, Liver Enzymes, and Fasting Blood Glucose in Patients with Non-alcoholic Fatty Liver Disease (NAFLD): A Randomized Controlled Trial.

Background: Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver disorders with a relatively high mortality rate. Berberine has recently been found to have some antidiabetic and antihyperlipidemic effects, although the evidence of its effectiveness in NAFLD is limited. To assess the efficacy of berberine among patients with NAFLD. Methods: The patients with NAFLD were randomly assigned to treatment with (n = 25) or without (n = 25) berberine. The patients in the intervention group took berberine 6.25 g per day and the control group had no berberine. All patients in the 2 groups had been recommended to have lifestyle training, including a low-fat diet and physical activity before randomization. Independent student t tests or Mann-Whitney U tests along paired t tests or Wilcoxon signed-rank tests were used. Analysis of covariance was also used to estimate the difference of the variables between the 2 groups adjusting for baseline characteristics. Results: The results indicated that berberine, compared with the control group, had no significant impact on lipid levels, including triglyceride (P = 0.350), total cholesterol (P = 0.120), high-density lipoproteins (P = 0.401), and low-density lipoproteins (P = 0.100). Similarly, no significant difference was observed between the treatment arms in the level of fasting blood glucose (P = 0.055) and liver enzymes, such as alkaline phosphatase (P = 0.109), serum glutamic-oxaloacetic transaminase (P = 0.366), and serum glutamic pyruvic transaminase (P = 0.436). The effect of berberine on body weight was also nonsignificant (P = 0.494) and even smaller than that of liver enzymes, with a mean difference of 1.8 kg (P = 0.304) in body weight. Conclusion: Berberine was not associated with a significant decrease in lipid profile, fasting blood glucose, or liver enzymes among patients with NAFLD.

19 F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.