One-step reverse transcriptase-free miRNA detection system and its application for detection of gastrointestinal cancers.

MicroRNAs (miRNAs) play pivotal roles in gene regulation and their dysregulation is implicated in various diseases, including cancer. Current methods for miRNA analysis often involve complex procedures and high costs, limiting their clinical utility. Therefore, there is a critical need for the development of simpler and more cost-effective miRNA detection techniques to enable early disease diagnosis. In this study, we introduce a novel one-enzyme for miRNA one-step detection method using Taq DNA polymerase, termed OSMOS-qPCR. We optimized the PCR buffer, PCR program, Taq DNA Polymerase concentrations and reverse PCR primer concentrations, resulted in a wide linear range from 100 fM to 0.001 fM (R2 > 0.98 for each miRNA), the detection limit for OSMOS-qPCR was 0.0025 fM. Furthermore, OSMOS-qPCR demonstrates excellent specificity to differentiation of less than 0.1 % nonspecific signal. Finally, we demonstrated the robust amplification efficiency, enabling the detection of trace amounts of cell-free miRNA in serum samples, and the excellent discrimination ability between gastrointestinal cancers and control subjects (AUC value = 1.0) if combined two miRNAs. The development of OSMOS-qPCR offering a simpler, cost-effective, and efficient detection method, has the potential to be non-invasive strategy for early detection of gastrointestinal cancers.

A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Identification and characterization of yellow stripe-like genes in maize suggest their roles in the uptake and transport of zinc and iron.

BACKGROUND: Yellow Stripe-Like (YSL) proteins are involved in the uptake and transport of metal ions. They play important roles in maintaining the zinc and iron homeostasis in Arabidopsis, rice (Oryza sativa), and barley (Hordeum vulgare). However, proteins in this family have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: In this study, we identified 19 ZmYSLs in the maize genome and analyzed their structural features. The results of a phylogenetic analysis showed that ZmYSLs are homologous to YSLs of Arabidopsis and rice, and these proteins are divided into four independent branches. Although their exons and introns have structural differences, the motif structure is relatively conserved. Analysis of the cis-regulatory elements in the promoters indicated that ZmYSLs might play a role in response to hypoxia and light. The results of RNA sequencing and quantitative real-time PCR analysis revealed that ZmYSLs are expressed in various tissues and respond differently to zinc and iron deficiency. The subcellular localization of ZmYSLs in the protoplast of maize mesophyll cells showed that they may function in the membrane system. CONCLUSIONS: This study provided important information for the further functional analysis of ZmYSL, especially in the spatio-temporal expression and adaptation to nutrient deficiency stress. Our findings provided important genes resources for the maize biofortification.

ZmGluTR1 is involved in chlorophyll biosynthesis and is essential for maize development.

Chlorophyll is the most important carrier of photosynthesis in plants and is therefore vital for plant growth and development. Synthesis of 5-aminolevulinic acid (ALA) is initiated and catalyzed by glutamyl-tRNA reductase (GluTR) and is the rate-limiting step in chlorophyll biosynthesis. GluTR is controlled by several regulating factors. Although many studies have investigated the structure and function of GluTR in plants, the maize (Zea mays L.) GluTR has not yet been reported. Here, we isolated and identified the first loss-of-function mutant of GluTR in plants from a maize mutagenic population. The stop-gain mutation in ZmGluTR1 resulted in leaf etiolation throughout the growing season. The level of intermediates of chlorophyll biosynthesis and photosynthetic pigments decreased markedly and abnormal chloroplast structure was also observed in the mutants. Further analysis revealed that the deletion of carboxyl terminal (C-terminal) led to premature transcription termination and this hindered the interaction with FLUORESCENT (FLU), thereby influencing the stability of mutated ZmGluTR1 and leading to abolish interaction with GluTR-binding protein (GluBP). Moreover, mutations in the catalytic domain or nicotinamide adenine dinucleotide phosphate (NADPH) binding domain were lethal under normal growth conditions. These results indicate that ZmGluTR1 plays a fundamental role in chlorophyll biosynthesis and maize development.

Real-Time Cell Temperature Fluctuation Monitoring System Using Precision Pt Sensors Coated with Low Thermal Capacity, Low Thermal Resistance, and Self-Assembled Multilayer Films.

Real-time monitoring of cell temperature fluctuation can help researchers better understand physiological phenomena and the effects of drug treatment on cells, which is a novel and important tool for cellular informatics. The platinum (Pt) temperature sensor is widely used in temperature measurement with the advantages of strong stability, great accuracy, and high sensitivity. However, the commercially available Pt sensors have large thermal resistance and heat capacity which are difficult to be applied for cell temperature measurement because only a very small amount of heat flux is generated by live cells. In this study, we designed a system using precision Pt thin-film temperature sensors with low heat capacity and thermal resistance. The Pt thin-film sensors are covered by a silicon nitride insulation layer grafted with a self-assembled multilayer silane film for promoting cell adhesion. The temperature coefficient of resistance of the Pt temperature sensor was about 2100 ppm/°C. The four-wire lead design next to the sensor detection area ensured maximum accuracy, resulting in a system noise below 0.01 °C over a long time. HEK-293T and HeLa cells were cultured on the sensor surface, respectively. The temperature fluctuation of 293T cells was monitored in a cell culture medium, showing a temperature increase of about 0.05-0.12 °C. The temperature fluctuation of HeLa cells treated with cisplatin was also measured and recorded, indicating a temperature decrease of 0.01 °C first and then a gradual temperature increase of 0.04 °C. The Pt sensor system we developed demonstrated high sensitivity and long stability for cell temperature fluctuation monitoring, which can be widely used in cell activity and cellular informatics studies.

Novel prospects for scarless wound healing: The roles of myofibroblasts and adipocytes.

Disturbances or defects in the process of wound repair can disrupt the delicate balance of cells and molecules necessary for complete wound healing, thus leading to chronic wounds or fibrotic scars. Myofibroblasts are one of the most important cells involved in fibrotic scars, and reprogramming provides a potential avenue to increase myofibroblast clearance. Although myofibroblasts have long been recognized as terminally differentiated cells, recent studies have shown that myofibroblasts have the capacity to be reprogrammed into adipocytes. This review intends to summarize the potential of reprogramming myofibroblasts into adipocytes. We will discuss myofibroblast lineage tracing, as well as the known mechanisms underlying adipocyte regeneration from myofibroblasts. In addition, we investigated different changes in myofibroblast gene expression, transcriptional regulators, signalling pathways and epigenetic regulators during skin wound healing. In the future, myofibroblast reprogramming in wound healing will be better understood and appreciated, which may provide new ideas for the treatment of scarless wound healing.

Paternal imprinting of dosage-effect defective1 contributes to seed weight xenia in maize.

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinted dosage-effect defective1 (ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5-10% seed weight reduction when ded1 is transmitted through the male, while homozygous mutants are defective with a 70-90% seed weight reduction. Ded1 encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

Dual-Action Icariin-Containing Thermosensitive Hydrogel for Wound Macrophage Polarization and Hair-Follicle Neogenesis.

Bone morphogenetic protein (BMP) pathway is essential for M2 macrophage polarization and hair-follicle neogenesis. Icariin, a flavonoid derived from Epimedium, is a mediator of the BMP pathway. Here, we develop a hydrogel formulation functionalized with icariin for regulation of macrophage polarization to accelerate wound healing and hair-follicle neogenesis. Compared to skin defects without icariin treatment, those treated with icariin+PEG hydrogel healed faster and had new hair follicles. Results in vivo showed that icariin+PEG hydrogel induced a higher level of M2 phenotypic transformation of macrophages. Moreover, icariin+PEG hydrogel significantly accelerated wound-repair process by reducing the invasion of inflammation, excessive deposition of collagen, immoderate activation of myofibroblasts, and increasing the regeneration of hair follicles. Furthermore, studies in vitro demonstrated that the icariin+PEG hydrogel induced macrophages to polarize to the M2 phenotype and dermal papilla cell to hair follicles. Finally, molecular analysis demonstrated that the icariin+PEG hydrogel increased the expression of BMP4 and Smad1/5 phosphorylation in skin wounds. These results demonstrate the therapeutic potential of icariin-containing thermosensitive hydrogels for inducing M2 macrophage polarization to accelerate wound healing and promote hair-follicle neogenesis by regulating the BMP pathway.

Durability Study of Thermal Transfer Printed Textile Electrodes for Wearable Electronic Applications.

Textile-based electronics hold great promise because they can endow wearable devices with soft and comfortable characteristics. However, the inherent porosity and fluffiness of fabrics result in high surface roughness, which presents great challenges in the manufacture of high-performance fabric electrodes. In this work, we propose a thermal transfer printing method to address the above challenges, in which electrodes or circuits of silver flake/thermoplastic polyurethane (TPU) composites are prefabricated on a release film by coating and laser engraving and then laminated by hot-pressing to a variety of fabrics and textiles. This universal and scalable production technique enables fabric electrodes to be made without compromising the original wearability, washability, and stretchability of textiles. The prepared fabric electrodes exhibit high conductivity (5.48 × 104 S/cm), high adhesion (≥1750 N/m), good abrasion/washing resistance, high patterning resolution (∼40 µm), and good electromechanical performance up to 50% strain. To demonstrate the potential applications, we developed textile-based radio frequency identification (RFID) tags for remote identification and a large-sized heater for wearable thermotherapy. More importantly, the solvent-free thermal transfer printing technology developed in this paper enables people to DIY interesting flexible electronics on clothes with daily tools, which can promote the commercial application of smart textile-based electronics.

OsHSD2 interaction with and phosphorylation by OsCPK21 is essential for lipid metabolism during rice caryopsis development.

Rice calcium-dependent protein kinase 21 (OsCPK21) is specifically and highly expressed throughout reproductive development and plays a critical role in rice pollen development by indirectly regulating the MIKC*-type MADS box transcription factor. However, little is known about the function of OsCPK21 in rice caryopsis development. In this study, we performed an in vitro pull-down experiment followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and identified hydroxysteroid dehydrogenase 2 (HSD2) as a candidate OsCPK21-interacting protein in 25 DAF (days after flowering) rice caryopses. Then, we verified the interaction between OsCPK21 and OsHSD2 using yeast two-hybrid and bimolecular fluorescence assays and revealed the in vitro phosphorylation of OsHSD2 by OsCPK21. Furthermore, oscpk21 and oshsd2 mutants were generated by the CRISPR/Cas9 technique, and we found that the lipid profiles were drastically changed in both oscpk21 and oshsd2, implying that OsHSD2 phosphorylated by OsCPK21 regulates lipid abundance in caryopsis development, thereby providing a potential target for the genetic improvement of rice grain quality in future lipid-related breeding and biotechnology applications.

The uPA System Differentially Alters Fibroblast Fate and Profibrotic Ability in Skin Fibrosis.

Skin fibrosis is a common pathological feature of various diseases, and few treatment strategies are available because of the molecular pathogenesis is poorly understood. The urokinase-type plasminogen activator (uPA) system is the major serine protease system, and its components uPA, urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1(PAI-1) are widely upregulated in fibrotic diseases, including hypertrophic scars, keloids, and scleroderma. Here, we found that the successful binding of uPA and uPAR activates the downstream peroxisome proliferator-activated receptor (PPAR) signalling pathway to reduce the proliferation, migration, and contraction of disease-derived fibroblasts, contributing to the alleviation of skin fibrosis. However, increased or robust upregulation of the inhibitor PAI-1 inhibits these effects, suggesting of the involvement of PAI-1 in skin fibrosis. Subsequent in vivo studies showed that uPAR inhibitors increased skin fibrosis in mouse models, while uPA agonists and PAI-1 inhibitors reversed these effects. Our findings demonstrate a novel role for the uPA system and highlights its relationships with skin fibrosis, thereby suggesting new therapeutic approaches targeting the uPA system.

Restriction of voltage decay by limiting low-voltage reduction in Li-rich oxide materials.

Li-rich layered oxides are recognized as promising candidates for next-generation Li-ion batteries owing to the high capacity of >250 mAh g-1, but the severe voltage fade has prevented their commercialization. It is widely known that high-voltage charge processes result in layered-to-spinel structural evolution and voltage fade in Li-rich layered oxides. This work emphasizes that limiting the low-voltage reduction can maintain the structure and voltage stability of Li-rich layered oxides after the 4.6 V high-voltage charge processes. A strategy of limiting the low-voltage (<2.8 V) reduction by cycling at 4.6-2.8 V was performed in traditional Li1.2Ni0.13Mn0.54Co0.13O2 and high-Ni Li1.2Ni0.222Mn0.504Co0.074O2. After 300 cycles, traditional Li1.2Ni0.13Mn0.54Co0.13O2 and high-Ni Li1.2Ni0.222Mn0.504Co0.074O2 cycling at 4.6-2 V showed midpoint discharge voltages of 2.83 V and 2.97 V with high voltage fade rates of 2.25 mV/cycle and 2.24 mV/cycle, respectively. While the two materials cycling at 4.6-2.8 V can maintain discharge midpoint voltages of 3.34 V and 3.49 V, with low voltage decay rates of 0.692 mV/cycle and 0.632 mV/cycle, respectively. To better understand the voltage performance, their electric structures were calculated by density functional theory. Physical characterizations were also used to analyze their differences in structural evolution. The results suggested that limiting low-voltage reduction in Li-rich layered oxides is highly necessary for maintaining their structure and voltage stability.

Mediation of Zinc and Iron Accumulation in Maize by ZmIRT2, a Novel Iron-Regulated Transporter.

Iron (Fe) is an essential micronutrient for plant growth. Iron-regulated transporters (IRTs) play important roles in Fe2+ uptake and transport in strategy I plants. Maize (Zea mays) belongs to a strategy II plant, in which mugineic acid (MA)-Fe3+ uptake is mainly carried out by Yellow Stripe 1 (YS1). However, ZmIRT1 was previously identified by our laboratory. In this study, we isolated a novel gene from maize (ZmIRT2), which is highly homologous to OsIRT2 and ZmIRT1. ZmIRT2 was expressed in roots and anther and was induced by Fe and zinc (Zn) deficiencies. ZmIRT2-GFP fusion protein localized to the plasma membrane and endoplasmic reticulum. ZmIRT2 reversed growth defects involving Zn and Fe uptake in mutant yeast. ZmIRT2 overexpression in maize led to elevated Zn and Fe levels in roots, shoots and seeds of transgenic plants. Transcript levels of ZmIRT1 were elevated in roots, while levels of YS1 were reduced in shoots of ZmIRT2 transgenic plants. Our results imply that ZmIRT2 may function solely with ZmIRT1 to mediate Fe uptake in roots. ZmIRT1, ZmIRT2 and ZmYS1 may function in a cooperative manner to maintain Zn and Fe homeostasis in ZmIRT2 overexpressing plants. Furthermore, ZmIRT2 could be used in fortification efforts to elevate Zn and Fe levels in crop plants.

Pentatricopeptide repeat protein CNS1 regulates maize mitochondrial complex III assembly and seed development.

Mitochondrial function relies on the assembly of electron transport chain complexes, which requires coordination between proteins encoded by the mitochondrion and those of the nucleus. Here, we cloned a maize (Zea mays) cytochrome c maturation FN stabilizer1 (CNS1) and found it encodes a pentatricopeptide repeat (PPR) protein. Members of the PPR family are widely distributed in plants and are associated with RNA metabolism in organelles. P-type PPR proteins play essential roles in stabilizing the 3'-end of RNA in mitochondria; whether a similar process exists for stabilizing the 5'-terminus of mitochondrial RNA remains unclear. The kernels of cns1 exhibited arrested embryo and endosperm development, whereas neither conventional splicing deficiency nor RNA editing difference in mitochondrial genes was observed. Instead, most of the ccmFN transcripts isolated from cns1 mutant plants were 5'-truncated and therefore lacked the start codon. Biochemical and molecular data demonstrated that CNS1 is a P-type PPR protein encoded by nuclear DNA and that it localizes to the mitochondrion. Also, one binding site of CNS1 located upstream of the start codon in the ccmFN transcript. Moreover, abnormal mitochondrial morphology and dramatic upregulation of alternative oxidase genes were observed in the mutant. Together, these results indicate that CNS1 is essential for reaching a suitable level of intact ccmFN transcripts through binding to the 5'-UTR of the RNAs and maintaining 5'-integrity, which is crucial for sustaining mitochondrial complex III function to ensure mitochondrial biogenesis and seed development in maize.

Maize Interveinal Chlorosis 1 links the Yang Cycle and Fe homeostasis through Nicotianamine biosynthesis.

The Yang cycle is involved in many essential metabolic pathways in plant growth and development. As extended products of the Yang cycle, the function and regulation network of ethylene and polyamines are well characterized. Nicotianamine (NA) is also a product of this cycle and works as a key metal chelator for iron (Fe) homeostasis in plants. However, interactions between the Yang cycle and NA biosynthesis remain unclear. Here, we cloned maize interveinal chlorosis 1 (mic1), encoding a 5'-methylthioadenosine nucleosidase (MTN), that is essential for 5'-methylthioadenosine (MTA) salvage and NA biosynthesis in maize (Zea mays). A single base G-A transition in the fourth exon of mic1 causes a Gly to Asp change, resulting in increased MTA, reduced Fe distribution, and growth retardation of seedlings. Knockout of ZmMIC1 but not its paralog ZmMTN2 by CRISPR/Cas9 causes interveinal chlorosis, indicating ZmMIC1 is mainly responsible for MTN activity in maize. Transcriptome analysis showed a typical response of Fe deficiency. However, metabolic analysis revealed dramatically reduced NA content in mic1, suggesting NA biosynthesis was impaired in the mutant. Exogenous application of NA transiently reversed the interveinal chlorosis phenotype of mic1 seedlings. Moreover, the mic1 mutant overexpressing a NA synthase gene not only recovered from interveinal chlorosis and growth retardation but was also fertile. These findings provide a link between the Yang cycle and NA biosynthesis, which highlights an aspect of Fe homeostasis regulation in maize.

Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis.

BACKGROUND: Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles. CONCLUSIONS: Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

Microplastics reduce the bioaccumulation and oxidative stress damage of triazole fungicides in fish.

Microplastics (MPs) and pesticides are typical representatives of harmful chemicals in polluted waters. It is understood that the combined toxicity may differ from that of a single toxic substance. Although their combined toxicities on aquatic organisms have practical significance and research value, they have received little attention due to their complicated interaction, and the mechanism has rarely been reported. In this paper, we designed a study to investigate the single and combined effects of polystyrene microplastics (PS-MPs) and the triazole fungicide difenoconazole on zebrafish, and to explore the mechanism of this effect. The results showed that PS-MPs could reduce the bioaccumulation of difenoconazole in zebrafish to a certain extent and alleviate the oxidative stress damage of difenoconazole in the zebrafish liver. The transcriptome and qRT-PCR data revealed the association of multiple pathways in the difenoconazole response, while the presence of PS-MPs ameliorated this effect in gene expression changes. Due to the properties of PS-MPs and the interaction between them, the toxic effect of difenoconazole when combined with PS-MPs is more prominent. These results provide a novel aspect to understand the environmental behavior of MPs and to evaluate the combined effect of MPs and pesticides on aquatic food.

ZmGRAS11, transactivated by Opaque2, positively regulates kernel size in maize.

Although the genetic basis for endosperm development in maize (Zea mays) has been well studied, the mechanism for coordinating grain filling with increasing kernel size remains elusive. Here, we report that increased kernel size was selected during modern breeding and identify a novel DELLA-like transcriptional regulator, ZmGRAS11, which positively regulates kernel size and kernel weight in maize. We find that Opaque2, a core transcription factor for zein protein and starch accumulation, transactivates the expression of ZmGRAS11. Our data suggest that the Opaque2-ZmGRAS11 module mediates synergistic endosperm enlargement with grain filling.

Metabolic engineering of astaxanthin-rich maize and its use in the production of biofortified eggs.

Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing ß-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.