p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR).

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

In vitro colonic fermentation characteristics of barley-koji differ from those of barley.

Barley-koji is prepared by inoculating barley, a beneficial prebiotic source, with the fungi Aspergillus luchuensis mut. kawachii. In this study, the prebiotic effects of barley-koji on human colonic microbiota were evaluated in vitro compared with barley, using pig feces. The enzyme-resistant fraction of the following sample groups each was added to respective fermenters: cellulose, barley (Commander and ß104), and barley-koji (Commander-koji and ß104-koji). Short-chain fatty acid and ammonia-nitrogen production increased and decreased, respectively, in barley-koji and barley groups. Furthermore, the propionate concentration increased in the barley group, showing a positive correlation with the abundance of the genus Dialister. In the barley-koji group, however, acetate and n-butyrate concentrations increased during the early stages of incubation, and the relative abundance of the genus Megasphaera was higher than those of the other genera. Therefore, this study demonstrated that barley-koji might possess beneficial physiological properties for colonic fermentation, which differ from those of barley.

KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics.

Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

Safety and efficacy of adzuki bean extract in subjects with moderate to high LDL-C: a randomized trial.

The safety and efficacy of polyphenol-containing adzuki bean extract on lipid metabolism were evaluated in human subjects in an 8-week, randomized, double-blind, placebo-controlled, parallel intervention study. No adverse effects were observed in the participants receiving adzuki bean extract. The adzuki bean group showed a significant increase in the ΔHDL-C concentration compared with the placebo group after 4 weeks of intervention (3.76 ± 7.79 mg/dL vs. -0.08 ± 6.03 mg/dL), respectively, and both groups showed reduced ∆HDL-C concentrations, with the adzuki bean extract group showing a return to the baseline levels (0.36 ± 5.36 mg/dL) and the placebo group showing a decrease to below the baseline levels (-3.17 ± 7.79 mg/dL) at week 8. This short-term study represents the first step in establishing the practicality, safety, and plausibility of HDL-C maintaining effects of adzuki bean extract in human subjects.

Effect of a combination of inulin and polyphenol-containing adzuki bean extract on intestinal fermentation in vitro and in vivo.

The effect of a combination of inulin (INU) and polyphenol-containing adzuki bean extract (AE) on intestinal fermentation was examined in vitro using fermenters for 48 h and in vivo using rats for 28 d. The total short-chain fatty acid concentrations in the fermenters were decreased by a combination of INU and AE, but the concentration in the INU + AE group was higher than the cellulose (CEL) and CEL + AE groups. The cecal propionate concentration was increased by a combination of INU and AE compared with their single supplement. The ammonia-nitrogen concentration in the fermenters and rat cecum was decreased by INU and AE. Cecal mucin levels were increased by INU and AE respectively. Therefore, our observations suggested that the combination of INU and AE might be a material of functional food that includes several healthy effects through intestinal fermentation.

Parallel measurement of dynamic changes in translation rates in single cells.

Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5' terminal oligopyrimidine (5' TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation.

Potato powders prepared by successive cooking-process depending on resistant starch content affect the intestinal fermentation in rats.

The effects of resistant starch (RS) in dry potato powders prepared by various processes on intestinal fermentation in rats were assessed. Rats were fed raw potato powder (RP), blanched potato powder (BP), steamed potato powder (SP), or drum-dried potato powder (DP) for 4 weeks. The cecal RS content was significantly higher in the RP group than in the control diet (CN) group and other dry potato powder groups. Cecum pH was significantly lower in the RP group compared to the CN group, and was also significantly lower than that in the SP, BP, and DP groups. Lactic acid bacteria levels in the RP group were significantly higher than those in the CN group, and levels in the SP group also increased relative to the control group. Lactobacillus levels in the RP group were higher than in the CN and other dry potato powder groups. Cecal short-chain fatty acid (SCFA) concentrations in the RP group followed by the SP group exhibited significantly higher levels relative to the control levels. Dry potato powders containing RS produced during the cooking process may represent a useful food material that increases intestinal concentrations of SCFA and enhances the growth of certain lactic acid bacteria.

Comparison of the effect of two types of whole mushroom (Agaricus bisporus) powders on intestinal fermentation in rats.

The effects of two types of mushroom (Agaricus bisporus; white, WM; brown, BM) powders on intestinal fermentation in rats were investigated in terms of the physical characteristics of animals and by bacterial and HPLC analyses of cecal contents. Short-chain fatty acid levels were found to be significantly higher in the WM group than in the BM and the control (CN) groups; coliform bacteria levels in the BM group were significantly lower than those in the CN group, with the WM group inducing an apparent but insignificant decrease in coliforms. Anaerobe levels in the WM group were significantly higher than those in the CN group and, compared with the CN group, the BM and WM groups exhibited significantly increased feces weight and cecum weight, respectively. These results indicate that the mushroom powders, and in particular the WM powder, have beneficial effects on the intestinal environment in rats.

Involvement of the vagus nerve and hepatic gene expression in serum adiponectin concentrations in mice.

Several humoral factors, such as adiponectin and urate, have been suggested to affect metabolic syndromes. Previously, we reported a reduction in blood adiponectin concentrations after a high-fructose diet partially via the vagus nerve in rats. Although a lithogenic diet (LD), i.e., supplementation of a normal control diet (CT) with 0.6% cholesterol and 0.2% sodium cholate, reduced blood adiponectin concentrations, the involvement of the vagus nerve in this mechanism remains unclear. To estimate the involvement of the vagus nerve in the regulation of blood adiponectin concentrations using an LD, male imprinting control region mice that had been vagotomized (HVx) or only laparotomized (Sham) were administered a CT or an LD for 10 weeks. Serum adiponectin concentrations in the Sham-LD, HVx-CT, and HVx-LD groups were reduced by half compared with the Sham-CT group. The hepatic mRNA levels of fibroblast growth factor 21 (Fgf21), which reportedly stimulates adiponectin secretion from white adipose tissue, were lower in the LD groups compared with the CT groups. HepG2 hepatoma cells showed that various bile acids reduced the mRNA expression of FGF21. Moreover, the LD increased serum urate concentrations and reduced hepatic expressions of the acyl-CoA oxidase 1 (Acox1) mRNA and glucokinase, suggesting insufficient regeneration of ATP from AMP. In conclusion, serum adiponectin concentration may be regulated via the vagus nerve in normal mice, whereas a reduction of hepatic Fgf21 mRNA by bile acids may also lower serum adiponectin levels. Moreover, the LD may promote hepatic AMP accumulation and subsequently increase the serum urate concentration in mice.

Temporal changes in the fermentation characteristics, bacterial community structure and the functionality of the predicted metagenome of a batch fermenter medium containing the upper gastrointestinal enzyme resistant fraction of white sorghum (Sorghum bicolor L. Moench).

Sorghum is a potential prebiotic ascribed to the high native resistant starch (RS) content. Our previous studies on raw sorghum have revealed prominent amino acid fermentation despite the high RS content. Interestingly, autoclaved-freeze-dried sorghum fed rats exhibited beneficial microbial and biochemical profiles. Having a keen interest to reciprocally scrutinize the underlying mechanisms behind these contrasting outcomes, we used an in vitro porcine batch fermentation model. The fermentable substrates in raw and autoclaved-freeze-dried (three cycles) sorghum (AC) after in vitro gastrointestinal digestion fostered similar bacterial community structures, yet with significant differences in the characteristic amylolytic microbial taxa abundance and their temporal variation. Further, significant differences in the concentration of organic acids in raw and AC manifested the differences in the predicted abundance of the underlying pathways of carbohydrate and organic acid metabolism. Thus, this study highlights the propensity of the heat-moisture treatment of sorghum in modifying the fermentability of its RS.

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL.

ABSTRACT: The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Inulin-type fructans with different degrees of polymerization improve lipid metabolism but not glucose metabolism in rats fed a high-fat diet under energy restriction.

BACKGROUND: Inulin-type fructan ameliorates metabolic diseases associated with obesity in animals. However, relatively little information is available on the comparative effects of inulins with different degree of polymerization (DP) on the lipid or glucose metabolism. AIM: The objective of this study was to investigate the effect of inulins with various DP on metabolic disorders associated with obesity in rats fed a high-fat diet under food restriction. METHODS: Rats were fed a high-fat diet supplemented with 5 % inulin-GR (Raftiline GR), inulin-Tokachi (Tokachi), or inulin-HP (Raftiline HP) without cellulose for 28 days at normal energy intakes or 14.5 % energy restriction. RESULTS: Under food restriction, the dietary inulin-Tokachi (mean DP 15) and -HP (mean DP 24), but not -GR (mean DP 10), reduced (p < 0.05) the serum cholesterol and triglyceride levels, and liver triglyceride concentration in rats, compared to the control diet. The cecal neutral steroid, bile acid, and propionate concentrations in the Tokachi and HP groups were higher (p < 0.05) than in the CONT group, and the cecal Bifidobacterium count in the Tokachi group was higher (p < 0.05) than in the other groups. CONCLUSIONS: Findings suggest that, depending on DP, dietary supplementation with inulin (DP 15 or DP 24) in rats fed a high-fat diet, regardless of food intake, positively modulates lipid metabolism and fecal microbiota but not glucose metabolism.

Effect of potato ethanol residue on rat plasma cholesterol levels.

We investigated the cholesterol-lowering effect of a potato ethanol residue (PER). The plasma cholesterol levels excluding high-density lipoprotein cholesterol were lower in the rats given a PER-containing diet for 6 weeks than in the control group, whereas the fecal cholesterol levels were higher. These results suggest that PER partially reduced plasma cholesterol levels via excretion of cholesterol into the feces.

Dietary L-cysteine improves the antioxidative potential and lipid metabolism in rats fed a normal diet.

L-cysteine works as a precursor of the antioxidant, glutathione. We investigated the effects of L-cysteine (1% and 2%) on lipid metabolism and the antioxidative system in rats fed a normal diet. Administering L-cysteine dependently decreased the food intake, fat mass weight and body weight dose. Dietary L-cysteine also decreased the triglyceride levels in the serum and liver. However, there were no significant differences in the hepatic TBARS and glutathione (GSH) levels among the groups. The activities of catalase and glutathione reductase in the rats receiving 2% L-cysteine were significantly higher (p<0.05) than in the control rats. These results suggest that dietary L-cysteine dose-dependently affected the antioxidative enzyme activities, and the lipid levels in the serum and liver which might be related to the reduced food intake.

Understanding the mobile healthcare applications continuance: The regulatory focus perspective.

BACKGROUND: Although mobile healthcare (mHealth) applications have proliferated and offer new opportunities for personal health management, many users exhibit discontinuance behavior. Various factors in mHealth applications can strongly impact users' continuance behavior, but it is not yet fully understood how they interact with each other to yield maximized effects. This study highlights the importance of identifying the antecedents and moderators of continuance intention to broaden the demographic reach of mHealth applications and thus contribute to maintaining a sustainable healthcare system. OBJECTIVE: This research explores three dimensions of perceived values (hedonic, utilitarian, and social) in mHealth platforms that lead to user satisfaction and, ultimately, continuance intention. It further investigates the moderating effects of personal traits defined by regulatory focus on the relationship between perceived value and user satisfaction. METHODS: Data was collected from 259 respondents with experience using the Samsung Health application. The research tests the proposed model and hypotheses by implementing PLS-SEM and multi-group analyses. RESULTS: Each dimension of perceived values positively influences user satisfaction, with hedonic and utilitarian values exhibiting stronger relationships. Regarding moderating effects, promotion (versus prevention) focus has a stronger enhancing effect on the positive relationship between utilitarian value and user satisfaction. In contrast, prevention (versus promotion) focus more strongly enhances the positive relationship between hedonic value and user satisfaction. Regulatory focus does not yield a significant moderating effect on the relationship between social value and user satisfaction. User satisfaction exerts a strong positive influence on continuance intention in mHealth environments. CONCLUSIONS: The moderating effect of individuals' regulatory focus has been identified. Combined effects of antecedents and moderators on user satisfaction influence their continuance intention in mHealth ecologies. Considering individual users' characteristics may guide mHealth application developers to design personalized platforms and establish enforced marketing strategies.

Gut microbiota-derived lipid metabolites facilitate regulatory T cell differentiation.

Commensal bacteria-derived metabolites are critical in regulating the host immune system. Although the impact of gut microbiota-derived hydrophilic metabolites, such as short-chain fatty acids, on immune cell functions and development has been well documented, the immunomodulatory effects of gut microbiota-derived lipids are still of interest. Here, we report that lipid extracts from the feces of specific-pathogen-free (SPF), but not germ-free (GF), mice showed regulatory T (Treg)-cell-inducing activity. We conducted RP-HPLC-based fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidome profiling and identified two bioactive lipids, 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and all-trans retinoic acid (atRA), with Treg-inducing activity in vitro. The luminal abundance of 9,10-DiHOME in the large intestine was significantly decreased by dextran sulfate sodium (DSS)-induced colitis, indicating that 9,10-DiHOME may be a potential biomarker of colitis. These observations implied that commensal bacteria-derived lipophilic metabolites might contribute to Treg development in the large intestine.

Japanese butterbur (Petasites japonicus) leaves increase hepatic oxidative stress in male rats.

We investigated the adverse effects of Japanese butterbur leaves (Petasites japonicus, Compositae) in male F344/DuCrj rats. The rats were fed a control diet or a treatment diet containing 5% butterbur leaf powder for 4 weeks. No differences were observed in body weight gain, food intake or feed efficiency between treatments, but relative liver weight in the butterbur group was significantly higher than that of the control group. In addition, thiobarbituric acid reactive substances (TBARs) and glutathione levels in the serum and liver of the butterbur group were higher than those of the control group. Hepatic glutathione reductase and glutathione S-transferase activities and mRNA expression in the butterbur leaf group were higher than in the control group. Furthermore, hepatic cytochrome 2E1 mRNA expression was higher than in the control group. In vitro, an acetone extract of the butterbur leaf powder showed the strongest increase in TBARs level in a hepatic homogenate through 4 d. Our findings suggest that feeding 5% butterbur leaf powder to rats can cause adverse effects by increasing oxidative stress.

Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade.

Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.