Gene Silencing Research Articles

Impact of KLF4, SHH, and Hif1a Knockdown on miRNA Expression in Malign Melanoma Cancer Stem Cells

Aim: microRNAs (miRNAs) play a pivotal role in gene regulation, influencing various cellular processes such as differentiation, proliferation, and apoptosis. This study investigated the expression of three specific miRNAs (Hsa-miR-21-5p, Hsa-miR-9-5p, and Hsa-miR-200a-5p) in malign melanoma stem cells (CSCs) and non-stem cells (NSCs). Method: CSCs and NCSCs were sorted from CHL-1 cells based on CD133 marker, a malignant melanoma cell line. CD133+ cells were treated with Hif1a, KLF4, and SHH siRNA and the expression levels of three different miRNAs were compared between groups. Results: Our results revealed that Hsa-miR-200a-5p expression was similar in both cell groups. Conversely, Hsa-miR-21-5p and Hsa-miR-9-5p were significantly upregulated in NCSCs. Further analysis showed that the knockdown of KLF4 did not significantly affect the expression levels of these miRNAs. However, silencing SHH resulted in a substantial downregulation of Hsa-miR-21-5p and a significant upregulation of Hsa-miR-9-5p. Additionally, Hif1a knockdown led to the downregulation of both Hsa-miR-21-5p and hsa-miR-9-5p. Conclusion: These findings highlight the complex regulatory mechanisms of miRNA expression in different cellular contexts and suggest potential roles for these miRNAs in response to specific gene silencing.

Phytol-induced interplant signaling in maize facilitates EXP-A20-driven resistance through ACO31-dependent ethylene accumulation against Ostrinia furnacalis.

Plants have evolved sophisticated defense mechanisms against insect herbivores, including cell wall fortification through lignin biosynthesis. Insect attack primes systemic acquired resistance in plants, preparing them to respond more swiftly and vigorously to subsequent insect assaults. Here, we found that Beauveria bassiana-exposed maize plants can emit phytol upon infestation by Spodoptera frugiperda, inducing plant-to-plant (PTP) communication of alert signals for neighboring plants, and revealed the expansin protein EXP-A20 as a pivotal node mediating maize defense responses in neighboring plants against the destructive pest Ostrinia furnacalis via stimulation of ethylene (ET) synthesis and lignin production. Through virus-induced gene silencing, we showed that EXP-A20 is essential for maize resistance, while downregulating ET and lignin pathways. Critically, protein-protein interactions determined via luciferase complementation and yeast two-hybrid assays demonstrated that EXP-A20 binds to and likely activates the ET-forming enzyme gene ACO31 to initiate defense signaling cascades, representing a novel signaling modality for expansins. Treatment with the plant volatile phytol has known insecticidal/priming activity, but we found that its effectiveness requires EXP-A20. This finding highlights the importance of EXP-A20 upstream of hormone-cell wall crosstalk in defense activation by volatiles. Overall, our multifaceted dissection of EXP-A20 revealed key molecular intersections underlying inducible maize immunity against herbivores. Furthermore, we provide functional evidence that extensive cell growth processes directly stimulate defense programs in plants. Our work opens new avenues for enhancing durable, broad-spectrum pest resistance in maize through the use of volatile organic compounds and PTP interactions.

A Virulence Factor from Sclerotinia sclerotiorum Targets the Host Chloroplast Proteins to Promote Infection

Chloroplasts are not only places for photosynthesis, but also participate in plant immunity and are important targets of pathogens. Pathogens secrete chloroplast-targeted proteins (CTPs) that disrupt host immunity and promote infection. Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus with a broad host range. However, little is known about the pathogenic mechanisms underlying this wide host range. In this study, we investigated the role of Chloroplast-Targeted Protein 1 (SsCTP1) secreted by S. sclerotiorum in pathogenesis, which inhibits plant immunity and promotes pathogen infections. SsCTP1 was highly up-regulated during the early stages of S. sclerotiorum infection in various hosts, and its transient expression in Nicotiana benthamiana revealed that it was predominantly localized within chloroplasts. Mutants with SsCTP1 deletion exhibited a similar growth rate and colony morphology to the wild type, but significantly reduced pathogenicity in various hosts. Moreover, SsCTP1 inhibited chitin-induced callose deposition and defense gene expression, and enhanced sensitivity to S. sclerotiorum in N. benthamiana. Similarly, transgenic Arabidopsis thaliana overexpressing SsCTP1 displayed an increased susceptibility to S. sclerotiorum. Furthermore, two host proteins that interact with SsCTP1, Coproporphyrinogen-III oxidase (GmCPX), and shikimate kinase 2 (GmSKL2) were identified by screening the soybean cDNA library, and these interactions were confirmed in vivo. Importantly, the silencing of NbCPX by virus-induced gene silencing enhanced N. benthamiana resistance to S. sclerotiorum. Our results indicate that SsCTP1 is an important pathogenic factor that contributes to the wide host range of S. sclerotiorum and may inhibit plant immunity by targeting the chloroplast proteins GmCPX and GmSKL2, which are ubiquitous in host plants.

Investigating the Expression and Function of HIF-1α in Neocaridina davidi During Embryo Cleavage Stage

Neocaridina davidi, a member of Decapoda within Crustacea, stands out due to its petite stature, robust reproductive capabilities and short molting cycle. Consequently, it has emerged as a valuable experimental model for investigations spanning ecology, development, physiology, and toxicology. Despite the pivotal role of Hypoxia Inducible Factor-1α (HIF-1α) in diverse physiological processes like biological hypoxia stress, cellular growth, and stress resistance, its functionality in crustaceans remains underexplored. Moreover, the role and function of HIF-1α in crustaceans, especially in embryonic stages, have been insufficiently addressed. This study delves into the function of HIF-1α during embryonic development. Initially, the complete sequence of the HIF-1α gene was acquired. Notably, the expression of HIF-1α during the cleavage stage surpassed that of subsequent phases. Subsequently, dsRNAHIF-1α was introduced into sexually mature shrimp, revealing that the inhibitory impact of dsRNA on gene expression in the parental generation could be inherited by the offspring, exerting a specific gene silencing effect in the embryo. The consequences of silencing HIF-1α in embryos manifested as varying degrees of premature division termination, besides, the glycolysis in the embryos was also affected by HIF-1α suppression. These results provide data support for understanding the early embryonic development of crustaceans and HIF-1α functions within it.

PfMORC protein regulates chromatin accessibility and transcriptional repression in the human malaria parasite, Plasmodium falciparum.

The environmental challenges the human malaria parasite, Plasmodium falciparum, faces during its progression into its various lifecycle stages warrant the use of effective and highly regulated access to chromatin for transcriptional regulation. Microrchidia (MORC) proteins have been implicated in DNA compaction and gene silencing across plant and animal kingdoms. Accumulating evidence has shed light on the role MORC protein plays as a transcriptional switch in apicomplexan parasites. In this study, using the CRISPR/Cas9 genome editing tool along with complementary molecular and genomics approaches, we demonstrate that PfMORC not only modulates chromatin structure and heterochromatin formation throughout the parasite erythrocytic cycle, but is also essential to the parasite survival. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments suggests that PfMORC binds to not only sub-telomeric regions and genes involved in antigenic variation but may also play a role in modulating stage transition. Protein knockdown experiments followed by chromatin conformation capture (Hi-C) studies indicate that downregulation of PfMORC impairs key histone marks and induces the collapse of the parasite heterochromatin structure leading to its death. All together these findings confirm that PfMORC plays a crucial role in chromatin structure and gene regulation, validating this factor as a strong candidate for novel antimalarial strategies.

Self-Assembly and Biological Properties of Highly Fluorinated Oligonucleotide Amphiphiles.

Nucleic acids, used as therapeutics to silence disease-related genes, offer significant advantages over small molecule drugs: they provide high specificity, the ability to target "undruggable" molecules, and adaptability to a wide range of disease phenotypes. However, their instability in biological media, as well their rapid clearance from the organism limit their applicability, necessitating the use of nanocarriers to overcome these challenges. Among these strategies, spherical nucleic acids (SNA) - composed of a densely packed corona of oligonucleotides around a nanoparticle - have emerged as a powerful tool, in particular when self-assembled from DNA amphiphiles. This non-covalent strategy however has caveats, especially when it comes to stability in complex biological media, where these SNAs disassemble in contact to serum proteins. Here, we developed highly fluorinated DNA amphiphiles that readily self-assemble into SNAs and have tunable stability profiles in biological media. They are made of branched fluorinated moieties with potentially improved biodegradability as compared to their linear counterparts. Depending on the number of fluorophilic interactions, the self-assembled SNAs can have excellent serum stabilities - up to days - and readily deliver nucleic acid therapeutics for gene silencing applications. These systems show great potential as promising candidates for nucleic acid-based therapies.

Preassembled complexes of hAgo2 and ssRNA delivered by nanoparticles: A novel silencing gene expression approach overcoming the absence of the canonical pathway of siRNA processing in the apicomplexan parasite Babesia microti, blood parasite of veterinary and zoonotic importance

Due to the lack of efficacy of the currently used chemical drugs, poor tick control, and lack of effective vaccines against Babesia, novel control strategies are urgently needed. In this regard, searching for anti-Babesia gene therapy may facilitate the control of this infection. Following this pattern, small interfering RNAs (siRNAs) are widely used to study gene function and hence open the way to control the parasite. However, the primary constraint of this approach is the lack of Babesia to RNA-induced silencing complex (RISC) enzymes, making siRNA impractical. In this study, we preassembled complexes with the human enzyme argonaute 2 (hAgo2) and a small interfering RNA (siRNA)/single-stranded RNA (ssRNA) against B. gibsoni and B. microti metabolite transporters. The assembled complexes were generated by developing a gene delivery system with chitosan dehydroascorbic acid nanoparticles. The delivery system effectively protected the loaded RNAi and targeted Babesia-infected RBCs with a relatively high internalization rate. The assembled complexes were successfully transfected into live parasites for specific slicing of Babesia targets. We demonstrated a reduction in the expression of target genes at the mRNA level. Furthermore, this silencing inhibited Babesia growth in vitro and in vivo. For the first time, we used this method to confirm the role of the assembled complexes in manipulating the noncanonical pathway of RNAi in Babesia parasites. This novel method provides a means of silencing Babesia genes to study their role in host-parasite interactions and as potential targets for gene therapy and control.

Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrPC, screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.

MiR-198 targets TOPORS: implications for oral squamous cell carcinoma pathogenesis

BackgroundmiRNAs play a critical role in the progression of various diseases, including oral squamous cell carcinoma (OSCC), which represents a major health concern and is one of the leading causes for new cancer cases worldwide. The miRNA dysregulation causes havoc and could be attributed to various factors, with epigenetic silencing of tumor suppressor genes being a major contributor to tumorigenesis. In this study, we have explored the tumor suppressive role of miR-198 in OSCC.MethodsThe tumor suppressive effect of miR-198 is established using miRNA analysis in OSCC cell lines, patient samples and xenograft nude mice model. The relationship between the miR-198 and TOPORS is explored using bioinformatics analyses, qRT-PCR, dual-luciferase reporter assay, Western blotting and cancer hall marks assays. The hypermethylation of the MIR198 promoter is confirmed using bisulfite sequencing PCR.ResultsWe have found miR-198 to be upregulated in OSCC cells treated with 5-Azacytidine, a known DNA methyltransferase inhibitor. Upregulation of miR-198 in 5-Azacytidine treated OSCC cells appears to be due to methylation of the MIR198 promoter. Using bioinformatics analysis and dual-luciferase reporter assay, we have identified TOPORS (TOP1 binding arginine/serine rich protein, E3 ubiquitin ligase) as a novel gene target for miR-198. miR-198-mediated repression of TOPORS decreases cell proliferation and anchorage-independent growth and enhances apoptosis of OSCC cells, which is dependent on the presence of the 3′UTR in TOPORS. An inverse correlation between the expression levels of miR-198 and TOPORS is observed in OSCC patient samples, highlighting the biological relevance of their interaction. Delivery of a synthetic miR-198 mimic to OSCC cells results in a significant decrease in xenograft size in nude mice, potentiating its use in therapeutics.ConclusionsThese results suggest that miR-198 is epigenetically silenced in OSCC, which promotes tumor growth, in part, by upregulating the levels of TOPORS.

Arsenic Trioxide (ATOIII) Induces NAD(P)H Quinone Oxidoreductase 1 (NQO1) Expression in Hepatic and Extrahepatic Tissues of C57BL/6 Mice.

Arsenic trioxide (ATOIII) has emerged as a potent therapeutic agent for acute promyelocytic leukemia (APL), yet its clinical application is often limited by significant adverse effects. This study investigates the molecular mechanisms underlying ATOIII's impact on cellular detoxification pathways, focusing on the regulation of NAD(P)H/quinone oxidoreductase (NQO1), a crucial enzyme in maintaining cellular homeostasis and cancer prevention. We explored ATOIII's effects on NQO1 expression in C57BL/6 mice and Hepa-1c1c7 cells, both independently and in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a known NQO1 inducer. Our findings revealed that ATOIII significantly increased NQO1 expression in hepatic and extrahepatic tissues, as well as in Hepa-1c1c7 cells, at mRNA, protein, and activity levels. This upregulation occurred both in the presence and absence of TCDD. Mechanistically, we demonstrated that ATOIII promotes the nuclear translocation of both nuclear factor erythroid 2-related factor-2 (NRF2) and aryl hydrocarbon receptor (AHR) transcription factors. Furthermore, ATOIII exposure increased antioxidant response element (ARE)-driven reporter gene activity, indicating a transcriptional mechanism of NQO1 induction. Notably, gene silencing experiments confirmed the critical roles of both NRF2 and AHR in mediating ATOIII-induced NQO1 expression. In conclusion, ATOIII exposure is found to upregulate the NQO1 enzyme through a transcriptional mechanism via AHR- and NRF2- dependent mechanisms, offering valuable insights into its therapeutic mechanisms.

Silencing CaPIP5K4-1 leads to decreased male fertility in Capsicum annuum L.

Phosphatidylinositol 4-phosphate 5-kinase gene CaPIP5K4-1 is highly expressed in the pepper anthers. Virus-induced gene silencing of CaPIP5K4-1 leads to reduced male fertility in pepper. The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a pivotal enzyme in the phosphatidylinositol signaling pathway, and its crucial involvement in both plant development and stress response has been established. Here, we found that the expression of CaPIP5K4-1 in pepper was significantly higher in the fertile flower buds compared to sterile flower buds. Furthermore, its expression was validated in anthers and pollens by qRT-PCR and RNA-ISH assays, respectively. Its GFP fusion protein was mainly located on the plasma membrane. Silencing CaPIP5K4-1 in fertile pepper accessions resulted in wrinkled pollen grain cell walls, decreased pollen germination efficiency, and inhibited pollen tube growth. The transcription levels of multiple genes in the phosphatidylinositol signaling pathway were also assessed. Five phospholipase C (PLC) genes were downregulated in silenced plants. On the contrary, inositol phosphatase SAC and phosphatase and tensin homolog (PTEN) were upregulated. This study reported the role of CaPIP5K4-1 in pepper male fertility and provided insights into the regulatory mechanisms of PI signaling in pepper.

Epigenetic Modifiers: Exploring the Roles of Histone Methyltransferases and Demethylases in Cancer and Neurodegeneration

Histone methyltransferases (HMTs) and histone demethylases (HDMs) are critical enzymes that regulate chromatin dynamics and gene expression through the addition and removal of methyl groups on histone proteins. HMTs, such as PRC2 and SETD2, are involved in the trimethylation of histone H3 at lysine 27 and lysine 36, influencing gene silencing and activation. Dysregulation of these enzymes often leads to abnormal gene expression and contributes to tumorigenesis. In contrast, HDMs including KDM7A and KDM2A reverse these methylation marks, and their dysfunction can drive disease progression. In cancer, the aberrant activity of specific HMTs and HDMs can lead to the silencing of tumor suppressor genes or the activation of oncogenes, facilitating tumor progression and resistance to therapy. Conversely, in neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), disruptions in histone methylation dynamics are associated with neuronal loss, altered gene expression, and disease progression. We aimed to comprehend the odd activity of HMTs and HDMs and how they contribute to disease pathogenesis, highlighting their potential as therapeutic targets. By advancing our understanding of these epigenetic regulators, this review provides new insights into their roles in cancer and neurodegenerative diseases, offering a foundation for future research.

Polyethylene glycol-phospholipid functionalized single-walled carbon nanotubes for enhanced siRNA systemic delivery

Small interfering RNAs (siRNA) technology has emerged as a promising therapeutic tool for human health conditions like cancer due to its ability to regulate gene silencing. Despite FDA-approved, their delivery remains localized and limiting their systemic use. This study used single-walled carbon nanotubes (SWNTs) functionalized with polyethylene glycolated (PEGylated) phospholipids (PL-PEG) derivatives for systemic siRNA delivery. We developed an siRNA systemic delivery vehicle (SWNT-siRNA) by conjugating SWNT functionalized with PL-PEG containing either amine (PA) or maleimide (MA). The functionalized SWNT with a lower molecular weight of PA produced the SWNT-siRNA conjugate system with the highest stability and high siRNA loading quantity. The system delivered siRNA to a panel of tumour cell lines of different organs (i.e. HeLa, H1299 and MCF-7) and a non-cancerous human embryonic kidney 293 cells (HEK293T) with high biocompatibility and low toxicity. The cellular uptake of SWNT-siRNA conjugates by epithelial cells was found to be energy dependent. Importantly, the presence of P-glycoprotein, a marker for drug resistance, did not inhibit SWNT-mediated siRNA delivery. Mouse xenograft model further confirmed the potential of SWNT-siRNA conjugates with a significant gene knock-down without signs of acute toxicity. These findings pave the way for potential gene therapy applications using SWNTs as delivery vehicles.

ArMYB89 and ArCOP1 interaction modulates anthocyanin biosynthesis in Acer rubrum leaves under low-temperature conditions.

Acer rubrum, a famous ornamental tree, produces bright red-coloured leaves because of the temperature decline from summer to autumn. This process's molecular mechanism is elusive, so we have investigated how anthocyanin biosynthesis is induced in A. rubrum leaves under low temperatures. The results of low-temperature treatment under light and dark conditions showed that the low-temperature promoted anthocyanin accumulation in A. rubrum is light-dependent. The transcriptome analysis showed that ArMYB89 was significantly highly expressed in leaves of A. rubrum growing under low temperatures with light conditions. The findings from the Dap-seq analysis, yeast one hybridisation, electrophoretic mobility shift assay and luciferase reporter assay indicated that the ArMYB89 transcription factor binds directly to the promoter of ArUGT52 and stimulates its transcription. The co-expression of ArUGT52 with ArMYB89 significantly induced anthocyanin levels under low temperatures with light conditions. Enzyme activity analysis showed that ArUGT52 could convert Cyanidins and Pelargonidins into Cyanidin-3-O-glucoside and Pelargonidin 3-glucoside, which are considered the main anthocyanins in red colour leaves of A. rubrum. The results of yeast two hybridisation, pulldown assay and bimolecular fluorescence complementation experiment showed an interaction between COP1 and ArMYB89, while in vivo and in vitro protein ubiquitination assay demonstrated that ArCOP1 ubiquitinates ArMYB89. Notably, co-expression of ArCOP1 with ArMYB89 significantly reduced anthocyanin levels, while the virus-induced gene silencing of ArCOP1 significantly induced anthocyanin levels under low temperatures with light conditions. In conclusion, this work revealed the molecular mechanism regulating anthocyanin accumulation in the A. rubrum leaves under low temperatures.

Repressor MrERF4 and Activator MrERF34 Synergistically Regulate High Flavonol Accumulation Under UV-B Irradiation in Morella rubra Leaves.

Flavonols are important plant photoprotectants to defence UV-B irradiation, however, the underlying transcriptional regulatory mechanism of rapid flavonol accumulation in response to UV-B remains unknown. In this study, content of flavonols was significantly induced from 0.11 to 3.80 mg/g fresh weight by UV-B irradiation in leaves of Morella rubra seedlings. MrERF34 was identified as an activator that can regulate the expression of MrFLS2, and promoted flavonol biosynthesis with activator MrMYB12 under UV-B treatment. Transient overexpression of MrERF34 resulted in higher flavonol accumulation, while virus-induced gene silencing of MrERF34 reduced the content of flavonols in bayberry leaves. We further demonstrated that a repressor MrERF4 inhibited the expression of MrERF34 and MrMYB12 as well as MrFLS2 via ERF-associated-amphiphilic repression motif. Exposure to UV-B reduced the promoter activity and transcription of MrERF4, which weakened the inhibitory effect of MrERF4 on MrERF34, MrMYB12, and MrFLS2, leading to a tremendous accumulation of flavonols. Such inhibitory roles of MrERF4 in regulation of flavonol biosynthesis were further validated by transient overexpression in leaves of Nicotiana benthamiana and M. rubra. These findings enrich the synergistical regulatory mechanisms between repressor and activators in flavonol biosynthesis, and provide new insights into photoprotectants biosynthesis to mitigate UV-B stress in plants.

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

Although shrimp are a valuable protein source, shrimp aquaculture has numerous challenges from various infectious diseases and understanding molecular mechanisms of disease pathogenesis is crucial for disease management. In this study, a gene-to-gene correlation network generated from a transcriptomic database of the stomach of shrimp infected with acute hepatopancreatic necrosis disease (AHPND) was used to identify a new α-arrestin, termed arrestin domain containing-3 gene (LvARRDC3), with crucial roles in development of both AHPND and white spot disease (WSD). Double stranded RNA-mediated silencing or plasmid-mediated overexpression of LvARRDC3 gene significantly decreased expression of WSSV genes (IE1, VP28, and ICP11) and viral genome copy numbers. Nevertheless, in AHPND, silencing the LvARRDC3 gene increased the AHPND-associated plasmid and Pir toxins copy numbers, whereas overexpression of LvARRDC3 had the opposite effect. An in vitro pathogen binding assay with recombinant LvARRDC3 protein produced robust binding to WSSV virions and AHPND-causing V. parahaemolyticus. Moreover, based on immunofluorescence, LvARRDC3 was localized in the cytoplasm of Spodoptera frugiperda (Sf9) insect cells. Therefore, we inferred that LvARRDC3 has a role in pathogen internalization, making it a valuable target for addressing AHPND and WSD and also a biomarker for marker-associated shrimp breeding.

The N-terminal polypeptide of a new shell matrix protein hicraqin accelerates the rate of calcium carbonate deposition.

Matrix proteins play important roles in shell formation by regulating the assembly of organic matrix and minerals. Here, we obtained a new matrix protein (hicraqin) from Hyriopsis cumingii. The amino acid sequence of hicraqin contains multiple aggregated (Gly)n (n > 2) residues, a feature unique to silk-like matrix proteins. In situ hybridization studies and tissue expression patterns demonstrated that hicraqin may be a prismatic layer matrix protein. In vitro experiments were performed using the peptide N-hicraqin (the N-terminal free sequence of hicraqin). In the in vitro crystallization of calcium carbonate, crystals resembling dumbbell, spindle, and lotus aragonite crystals were observed under scanning electron microscopy and confirmed as calcite by Raman spectroscopy. In the in vitro crystallization system of calcium carbonate with the addition of magnesium ions, aragonite plates were generated with 50 μg/mL of the peptide N-hicraqin. The fluorescent labeling analysis indicated that N-hicraqin was involved in the crystallization process. The crystallization rate experiment showed that the peptide N-hicraqin plays a role in promoting crystallization. Following the silencing of the hicraqin gene by RNA interference, its expression was reduced by about 61%. There was incomplete formation of the organic framework outside the prismatic layer. Overall, the present study showed that N-hicraqin participates in the crystallization process and acts as a framework protein that influences the formation of the organic framework of the prismatic layer.

GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-β (lycopene β-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas. SynopsisLycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

A Computational Approach for Designing and Validating Small Interfering RNA against SARS-CoV-2 Variants.

The aim of this study is to develop a novel antiviral strategy capable of efficiently targeting a broad set of SARS-CoV-2 variants. Since the first emergence of SARS-CoV-2, it has rapidly transformed into a global pandemic, posing an unprecedented threat to public health. SARS-CoV-2 is prone to mutation and continues to evolve, leading to the emergence of new variants capable of escaping immune protection achieved due to previous SARS-CoV-2 infections or by vaccination. RNA interference (RNAi) is a remarkable biological mechanism that can induce gene silencing by targeting complementary mRNA and inhibiting its translation. In this study, using the computational approach, we predicted the most efficient siRNA capable of inhibiting SARS-CoV-2 variants of concern (VoCs). The presented siRNA was characterized and evaluated for its thermodynamic properties, offsite-target hits, and in silico validation by molecular docking and molecular dynamics simulations (MD) with Human AGO2 protein. The study contributes to the possibility of designing and developing an effective response strategy against existing variants of concerns and preventing further.

BSA-seq and transcriptome analysis reveal GhDSA05 as a candidate gene responsible for sensitivity to defoliants in Gossypium hirsutum L.

With the development of machine-harvested cotton, the application of defoliants is a key technology that can promote defoliation and reduce impurities in cotton fibers. Therefore, sensitivity to defoliants is a very important trait in machine-harvested cotton. In this study, to uncover the genetic basis of defoliant sensitivity in cotton, bulked segregant analysis by sequencing was used with a F2 population derived from Xinluzao 57 (XLZ57, defoliant sensitive) and Nan 2 (N2, defoliant insensitive) to mapped the quantitative trait locus (QTL) to a 3.66-Mb interval at chromosome A05. Then, 284 F2 individuals were used to validate and narrow the QTL to a 1.6-Mb interval and was named qSD-A05. To identify the candidate genes, dynamic transcriptome analysis of the abscission zone (AZ) in XLZ57 and N2 after defoliant treatment was performed. A total of 132 and 142 common differentially expressed genes (DEGs) were identified at all time points in N2 and XLZ57, respectively, and were enriched in the defense response pathway and abscisic acid-activated signaling pathway. More importantly, 15 DEGs were identified in the qSD-A05 interval, among which, Ghi_A05G23601 was significantly upregulated after defoliant treatment. There were multiple variants between N2 and XLZ57, including missense and frameshift mutations. Therefore, Ghi_A05G23601 was regarded as the key candidate gene and was named GhDSA05, which encodes a Facilitator superfamily transporter protein. To verify the function of GhDSA05, virus-induced gene silencing was performed, which showed that the GhDSA05-silenced plants exhibited decreased defoliant sensitivity. Additionally, the expression of organ abscission-related genes was downregulated in the GhDSA05-silenced plants. In summary, GhDSA05 positively regulates the formation of AZ under defoliant treatment and promotes leaf abscission in cotton. The results of this study not only enhance our knowledge of the defoliation mechanism of cotton but also provide a target for the genetic improvement of defoliant sensitivity.