Mitochondria-associated endoplasmic reticulum membrane (MAM) is a structural link between mitochondria and endoplasmic reticulum (ER). MAM regulates Ca
transport from the ER to mitochondria via an ...IP3R1-GRP75-VDAC1 complex-dependent mechanism. Excessive MAM formation may cause mitochondrial Ca
overload and mitochondrial dysfunction. However, the exact implication of MAM formation in metabolic syndromes remains debatable. Here, we demonstrate that PDK4 interacts with and stabilizes the IP3R1-GRP75-VDAC1 complex at the MAM interface. Obesity-induced increase in PDK4 activity augments MAM formation and suppresses insulin signaling. Conversely, PDK4 inhibition dampens MAM formation and improves insulin signaling by preventing MAM-induced mitochondrial Ca
accumulation, mitochondrial dysfunction, and ER stress. Furthermore,
mice exhibit reduced MAM formation and are protected against diet-induced skeletal muscle insulin resistance. Finally, forced formation and stabilization of MAMs with synthetic ER-mitochondria linker prevented the beneficial effects of PDK4 deficiency on insulin signaling. Overall, our findings demonstrate a critical mediatory role of PDK4 in the development of skeletal muscle insulin resistance via enhancement of MAM formation.
Human society has become increasingly reliant on plastic because it allows for convenient and sanitary living. However, recycling rates are currently low, which means that the majority of plastic ...waste ends up in landfills or the ocean. Increasing recycling and upcycling rates is a critical strategy for addressing the issues caused by plastic pollution, but there are several technical limitations to overcome. This article reviews advancements in polymer technology that aim to improve the efficiency of recycling and upcycling plastic waste. In food packaging, natural polymers with excellent gas barrier properties and self-cleaning abilities have been introduced as environmentally friendly alternatives to existing materials and to reduce food-derived contamination. Upcycling and valorization approaches have emerged to transform plastic waste into high-value-added products. Recent advancements in the development of recyclable high-performance plastics include the design of super engineering thermoplastics and engineering chemical bonds of thermosets to make them recyclable and biodegradable. Further research is needed to develop more cost-effective and scalable technologies to address the plastic pollution problem through sustainable recycling and upcycling.
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•Summarized recent advances for increasing plastic recycling/upcycling efficiency.•Reviewed technologies for converting plastic waste into other polymer materials.•Materials with higher performance than the input plastic waste can be manufactured.
Rice is an important cereal crop worldwide, the growth of which is affected by rice blast disease, caused by the fungal pathogen
. As climate change increases the diversity of pathogens, the disease ...resistance genes (
genes) in plants must be identified. The major blast-resistance genes have been identified in
rice varieties; therefore,
rice varieties with
genes now need to be identified. Because leucine-rich repeat (LRR) domain proteins possess
-gene properties, we used bioinformatics analysis to identify the rice candidate LRR domain receptor-like proteins (OsLRR-RLPs).
, which contains six LRR domains, showed differences in the DNA sequence, containing 43 single-nucleotide polymorphisms (SNPs) in
and
subpopulations. The results of the
inoculation analysis indicated that
varieties with partial deletion of
showed susceptibility, whereas
varieties with intact
showed resistance. The
mutant, generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), showed increased pathogen susceptibility, whereas plants overexpressing this gene showed pathogen resistance. These results indicate that
confers resistance to rice, and
may be useful for breeding resistant cultivars.
Targeted deep sequencing across broad genomic regions has been used to detect circulating tumor DNA (ctDNA) in pancreatic ductal adenocarcinoma (PDAC) patients. However, since most PDACs harbor a ...mutation in KRAS, sequencing of broad regions needs to be systemically compared to analyzing only KRAS mutations for PDAC. Using capture-based targeted deep sequencing, we detected somatic tumor mutations in 17 fine needle aspiration biopsy and 69 longitudinal cell-free DNA (cfDNA) samples from 17 PDAC patients. KRAS mutations were detected in 10 out of 17 pretreatment patient plasma samples. Next, interrogation of genetic alterations in matched primary tumor samples detected ctDNA in 12 of 17 pretreatment plasma samples and cfDNA sequencing across the 83 target genes identified ctDNA in 15 of 17 cases (88.2% sensitivity). This improved sensitivity of ctDNA detection resulted in enhanced tumor burden monitoring when we analyzed longitudinal plasma samples. We found that cfDNA sequencing detected the lowest mutant allelic fractions and number of variants when complete response or partial response to chemotherapy was achieved. We demonstrated that ctDNA levels measured by targeted deep sequencing sensitively indicate the presence of cancer and correlate well with clinical responses to therapy and disease progression in PDAC patients.
Head and neck squamous cell carcinoma (HNSCC) undergoes stepwise progression from normal tissues to precancerous leukoplakia, primary HNSCC, and metastasized tumors. To delineate the heterogeneity of ...tumor cells and their interactions during the progression of HNSCC, we employ single-cell RNA-seq profiling for normal to metastasized tumors. We can identify the carcinoma in situ cells in leukoplakia lesions that are not detected by pathological examination. In addition, we identify the cell type subsets of the Galectin 7B (LGALS7B)-expressing malignant cells and CXCL8-expressing fibroblasts, demonstrating that their abundance in tumor tissue is associated with unfavorable prognostic outcomes. We also demonstrate the interdependent ligand-receptor interaction of COL1A1 and CD44 between fibroblasts and malignant cells, facilitating HNSCC progression. Furthermore, we report that the regulatory T cells in leukoplakia and HNSCC tissues express LAIR2, providing a favorable environment for tumor growth. Taken together, our results update the pathobiological insights into cell-cell interactions during the stepwise progression of HNSCCs.
Targeted deep sequencing is increasingly used to detect low-allelic fraction variants; it is therefore essential that errors that constitute baseline noise and impose a practical limit on detection ...are characterized. In the present study, we systematically evaluate the extent to which errors are incurred during specific steps of the capture-based targeted sequencing process.
We removed most sequencing artifacts by filtering out low-quality bases and then analyze the remaining background noise. By recognizing that plasma DNA is naturally fragmented to be of a size comparable to that of mono-nucleosomal DNA, we were able to identify and characterize errors that are specifically associated with acoustic shearing. Two-thirds of C:G > A:T errors and one quarter of C:G > G:C errors were attributed to the oxidation of guanine during acoustic shearing, and this was further validated by comparative experiments conducted under different shearing conditions. The acoustic shearing step also causes A > G and A > T substitutions localized to the end bases of sheared DNA fragments, indicating a probable association of these errors with DNA breakage. Finally, the hybrid selection step contributes to one-third of the remaining C:G > A:T and one-fifth of the C > T errors.
The results of this study provide a comprehensive summary of various errors incurred during targeted deep sequencing, and their underlying causes. This information will be invaluable to drive technical improvements in this sequencing method, and may increase the future usage of targeted deep sequencing methods for low-allelic fraction variant detection.
Magnetically actuated soft robots may improve the treatment of disseminated intravascular coagulation. Significant progress has been made in the development of soft robotic systems that steer ...catheters. A more challenging task, however, is the development of systems that steer sub-millimeter-diameter guidewires during intravascular treatments; a novel microrobotic approach is required for steering. In this article, we develop a novel, magnetically actuated, soft microrobotic system, increasing the steerability of a conventional guidewire. The soft microrobot is attached to the tip of the guidewire, and it is magnetically steered by changing the direction and intensity of an external magnetic field. The microrobot is fabricated via replica molding and features a soft body made of polydimethylsiloxane, two permanent magnets, and a microspring. We developed a mathematical model mapping deformation of the soft microrobot using a feed-forward approach toward steering. Then, we used the model to steer a guidewire. The angulation of the microrobot can be controlled from 21.1° to 132.7° by using a magnetic field of an intensity of 15 mT. Steerability was confirmed by two-dimensional
in vitro
tracking. Finally, a guidewire with the soft microrobot was tested by using a three-dimensional (3D) phantom of the coronary artery to verify steerability in 3D space.
Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. ...However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.
Accurate detection of genomic alterations using high-throughput sequencing is an essential component of precision cancer medicine. We characterize the variant allele fractions (VAFs) of somatic ...single nucleotide variants and indels across 5095 clinical samples profiled using a custom panel, CancerSCAN. Our results demonstrate that a significant fraction of clinically actionable variants have low VAFs, often due to low tumor purity and treatment-induced mutations. The percentages of mutations under 5% VAF across hotspots in EGFR, KRAS, PIK3CA, and BRAF are 16%, 11%, 12%, and 10%, respectively, with 24% for EGFR T790M and 17% for PIK3CA E545. For clinical relevance, we describe two patients for whom targeted therapy achieved remission despite low VAF mutations. We also characterize the read depths necessary to achieve sensitivity and specificity comparable to current laboratory assays. These results show that capturing low VAF mutations at hotspots by sufficient sequencing coverage and carefully tuned algorithms is imperative for a clinical assay.
Clinical prescription of cisplatin, one of the most widely used chemotherapeutic agents, is limited by its side effects, particularly tubular injury–associated nephrotoxicity. Since details of the ...underlying mechanisms are not fully understood, we investigated the role of pyruvate dehydrogenase kinase (PDK) in cisplatin-induced acute kidney injury. Among the PDK isoforms, PDK4 mRNA and protein levels were markedly increased in the kidneys of mice treated with cisplatin, and c-Jun N-terminal kinase activation was involved in cisplatin-induced renal PDK4 expression. Treatment with the PDK inhibitor sodium dichloroacetate (DCA) or genetic knockout of PDK4 attenuated the signs of cisplatin-induced acute kidney injury, including apoptotic morphology of the kidney tubules along with numbers of TUNEL-positive cells, cleaved caspase-3, and renal tubular injury markers. Cisplatin-induced suppression of the mitochondrial membrane potential, oxygen consumption rate, expression of electron transport chain components, cytochrome c oxidase activity, and disruption of mitochondrial morphology were noticeably improved in the kidneys of DCA-treated or PDK4 knockout mice. Additionally, levels of the oxidative stress marker 4-hydroxynonenal and mitochondrial reactive oxygen species were attenuated, whereas superoxide dismutase 2 and catalase expression and glutathione synthetase and glutathione levels were recovered in DCA-treated or PDK4 knockout mice. Interestingly, lipid accumulation was considerably attenuated in DCA-treated or PDK4 knockout mice via recovered expression of peroxisome proliferator-activated receptor-α and coactivator PGC-1α, which was accompanied by recovery of mitochondrial biogenesis. Thus, PDK4 mediates cisplatin-induced acute kidney injury, suggesting that PDK4 might be a therapeutic target for attenuating cisplatin-induced acute kidney injury.
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