Nutrient metabolism in regulating intestinal stem cell homeostasis.

Intestinal stem cells (ISCs) are known for their remarkable proliferative capacity, making them one of the most active cell populations in the body. However, a high turnover rate of intestinal epithelium raises the likelihood of dysregulated homeostasis, which is known to cause various diseases, including cancer. Maintaining precise control over the homeostasis of ISCs is crucial to preserve the intestinal epithelium's integrity during homeostasis or stressed conditions. Recent research has indicated that nutrients and metabolic pathways can extensively modulate the fate of ISCs. This review will explore recent findings concerning the influence of various nutrients, including lipids, carbohydrates, and vitamin D, on the delicate balance between ISC proliferation and differentiation.

Membrane phospholipid remodeling modulates nonalcoholic steatohepatitis progression by regulating mitochondrial homeostasis.

BACKGROUND AND AIMS: NASH, characterized by inflammation and fibrosis, is emerging as a leading etiology of HCC. Lipidomics analyses in the liver have shown that the levels of polyunsaturated phosphatidylcholine (PC) are decreased in patients with NASH, but the roles of membrane PC composition in the pathogenesis of NASH have not been investigated. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), a phospholipid (PL) remodeling enzyme that produces polyunsaturated PLs, is a major determinant of membrane PC content in the liver. APPROACH AND RESULTS: The expression of LPCAT3 and the correlation between its expression and NASH severity were analyzed in human patient samples. We examined the effect of Lpcat3 deficiency on NASH progression using Lpcat3 liver-specific knockout (LKO) mice. RNA sequencing, lipidomics, and metabolomics were performed in liver samples. Primary hepatocytes and hepatic cell lines were used for in vitro analyses. We showed that LPCAT3 was dramatically suppressed in human NASH livers, and its expression was inversely correlated with NAFLD activity score and fibrosis stage. Loss of Lpcat3 in mouse liver promotes both spontaneous and diet-induced NASH/HCC. Mechanistically, Lpcat3 deficiency enhances reactive oxygen species production due to impaired mitochondrial homeostasis. Loss of Lpcat3 increases inner mitochondrial membrane PL saturation and elevates stress-induced autophagy, resulting in reduced mitochondrial content and increased fragmentation. Furthermore, overexpression of Lpcat3 in the liver ameliorates inflammation and fibrosis of NASH. CONCLUSIONS: These results demonstrate that membrane PL composition modulates the progression of NASH and that manipulating LPCAT3 expression could be an effective therapeutic for NASH.

Targeting phospholipid remodeling pathway improves insulin resistance in diabetic mouse models.

Previous studies have revealed that membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) is involved in the development of insulin resistance in type 2 diabetes. In this study, we aimed to investigate the therapeutic potential of targeting Lpcat3 in the treatment of insulin resistance in diabetic mouse models. Lpcat3 expression was suppressed in the whole body by antisense oligonucleotides (ASO) injection or in the liver by adeno-associated virus (AAV)-encoded Cre in high-fat diet (HFD)-induced and genetic ob/ob type 2 diabetic mouse models. Glucose tolerance test (GTT), insulin tolerance test (ITT), fasting blood glucose, and insulin levels were used to assess insulin sensitivity. Lipid levels in the liver and serum were measured. The expression of genes involved in de novo lipogenesis was analyzed by real-time RT-PCR. Metabolic rates were measured by indirect calorimetry using the Comprehensive Lab Animal Monitoring System (CLAMS). Our data demonstrate that acute knockout of hepatic Lpcat3 by AAV-Cre improves both hyperglycemia and hypertriglyceridemia in HFD-fed mice. Similarly, whole-body ablation of Lpcat3 by ASO administration improves obesity and insulin resistance in both HFD-fed and ob/ob mice. These findings demonstrate that targeting LPCAT3 could be a novel therapy for insulin resistance.

Ultrasonic microwave-assisted extraction coupled with macroporous resin chromatography for the purification of antioxidant phenolics from waste jackfruit (Artocarpus heterophyllus Lam.) peels.

An efficient ultrasonic microwave-assisted extraction (UMAE) coupled with macroporous resin chromatography technique was successfully used for the extraction and purification of antioxidant phenolics from jackfruit by-products (peels). After optimization by single factor experiments and response surface methodology, the optimum extraction conditions for UMAE were: ethanol concentration 63%, solvent-to-solid ratio 34 mL/g, microwave power 160 W and irradiation time 20 min. Under the optimal condition, the phenolics extraction yield was 8.14 mg GAE/g DW. After the purification by macroporous resin AB-8, the purity of antioxidant phenolics from UMAE extracts improved from 13.59 to 49.07%. Furthermore, ABTS radical scavenging activities were also significantly increased from 35.95 ± 2.21 to 162.36 ± 10.26 mg TE/g. HPLC analysis revealed that gallic acid, chlorogenic acid, and catechin were three dominant antioxidant phenolics in jackfruit peels. All of the results demonstrated that waste jackfruit peels could be utilized as a good source of phenolics with strong antioxidant activities in food and pharmaceutical industry.

AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice.

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7+ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7+ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin+ cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.

Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C-C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism.

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection.

MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce "contaminative" nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide-PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.

Nanolock-Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue.

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock-nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock-nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.

Sequence-Specific Covalent Capture Coupled with High-Contrast Nanopore Detection of a Disease-Derived Nucleic Acid Sequence.

Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence-specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast-in essence, digital-signal that enables sensitive, single-molecule sensing of the cross-linked duplexes.