A Method to Quantitatively Examine Heat Stress-Induced Alternative Splicing in Plants by RNA-Seq and RT-PCR.

Alternative splicing (AS) of pre-mRNAs is a type of post-transcriptional regulation in eukaryotes that expands the number of mRNA isoforms. Intron retention is the primary form of AS in plants and occurs more frequently when plants are exposed to environmental stresses. Several wet-lab and bioinformatics techniques are used to detect AS events, but these techniques are technically challenging or unsuitable for studying AS in plants. Here, we report a method that combines RNA-sequencing and reverse transcription PCR for visualizing and validating heat stress-induced AS events in plants, using Arabidopsis thaliana and HEAT SHOCK PROTEIN21 (HSP21) as examples.

Viperin-like proteins interfere with RNA viruses in plants.

Plant viruses cause substantial losses in crop yield and quality; therefore, devising new, robust strategies to counter viral infections has important implications for agriculture. Virus inhibitory protein endoplasmic reticulum-associated interferon-inducible (Viperin) proteins are conserved antiviral proteins. Here, we identified a set of Viperin and Viperin-like proteins from multiple species and tested whether they could interfere with RNA viruses in planta. Our data from transient and stable overexpression of these proteins in Nicotiana benthamiana reveal varying levels of interference against the RNA viruses tobacco mosaic virus (TMV), turnip mosaic virus (TuMV), and potato virus x (PVX). Harnessing the potential of these proteins represents a novel avenue in plant antiviral approaches, offering a broader and more effective spectrum for application in plant biotechnology and agriculture. Identifying these proteins opens new avenues for engineering a broad range of resistance to protect crop plants against viral pathogens.

Molecular farming for sustainable production of clinical-grade antimicrobial peptides.

Antimicrobial peptides (AMPs) are emerging as next-generation therapeutics due to their broad-spectrum activity against drug-resistant bacterial strains and their ability to eradicate biofilms, modulate immune responses, exert anti-inflammatory effects and improve disease management. They are produced through solid-phase peptide synthesis or in bacterial or yeast cells. Molecular farming, i.e. the production of biologics in plants, offers a low-cost, non-toxic, scalable and simple alternative platform to produce AMPs at a sustainable cost. In this review, we discuss the advantages of molecular farming for producing clinical-grade AMPs, advances in expression and purification systems and the cost advantage for industrial-scale production. We further review how 'green' production is filling the sustainability gap, streamlining patent and regulatory approvals and enabling successful clinical translations that demonstrate the future potential of AMPs produced by molecular farming. Finally, we discuss the regulatory challenges that need to be addressed to fully realize the potential of molecular farming-based AMP production for therapeutics.

Prophylactic and Therapeutic Efficacy of Ultrasonicated Rosmarinus officinalis Ethanolic Extract and its Chitosan-Loaded Nanoparticles Against Eimeria tenella Infected Broiler Chickens.

PURPOSE: The in vivo efficacy of ultrasonicated Rosmarinus officinalis ethanolic extract (UROEE) and its chitosan-loaded nanoparticles (UROEE-CsNPs) was investigated as a dietary prophylactic agent and as a therapeutic treatment against Eimeria tenella infected broiler chickens. METHODS: Chickens were infected with 4 × 104 E. tenella oocysts at 21 days old for primary infection and with 8 × 104 oocysts at 35 days old for secondary infection. Eleven experimental groups were conducted. Dietary addition of 100 mg/kg UROEE and 20 mg/kg for CsNPs as well as UROEE-CsNPs were included for prophylactic groups from day 1 to 42. The same doses were used for therapeutic treatment groups for 5 constitutive days. Oocyst output in feces was counted. Histopathological and immunohistochemical studies were conducted. Gene expression of pro-inflammatory cytokines as IFN-γ, IL-1ß and IL-6 as well as anti-inflammatory cytokines as IL-10 and TGF-ß4 was analyzed using semi-quantitative reverse transcriptase-PCR. RESULTS: The results showed an efficacy of UROEE, CsNPs and UROEE-CsNPs in reduction of oocyst excretion and improving the cecal tissue architecture. CD4+ and CD8+ T lymphocytes protein expression were reduced. E. tenella infection lead to upregulation of pro-inflammatory cytokines as IFN-γ, IL-1ß, IL-6 and anti-inflammatory cytokines as TGF-ß4 following primary infection, while their expression was downregulated following secondary infection. CONCLUSION: The dietary prophylactic additives and therapeutic treatments with UROEE, CsNPs and UROEE-CsNPs could decrease the inflammatory response to E. tenella as indicated by oocyst output reduction, histopathological improvements, CD4+ and CD8+ T cells protein expression reduction as well as reducing mRNA expression levels of the tested cytokines following primary and secondary infections. Consequently, these results will help to develop better-combating strategies for the control and prevention of coccidiosis on poultry farms as a dietary prophylactic agent or as a therapeutic treatment.

Crop bioengineering via gene editing: reshaping the future of agriculture.

Genome-editing technologies have revolutionized research in plant biology, with major implications for agriculture and worldwide food security, particularly in the face of challenges such as climate change and increasing human populations. Among these technologies, clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated protein [Cas] systems are now widely used for editing crop plant genomes. In this review, we provide an overview of CRISPR-Cas technology and its most significant applications for improving crop sustainability. We also review current and potential technological advances that will aid in the future breeding of crops to enhance food security worldwide. Finally, we discuss the obstacles and challenges that must be overcome to realize the maximum potential of genome-editing technologies for future crop and food production.

Multitrait engineering of Hassawi red rice for sustainable cultivation.

Sustainable agriculture requires locally adapted varieties that produce nutritious food with limited agricultural inputs. Genome engineering represents a viable approach to develop cultivars that fulfill these criteria. For example, the red Hassawi rice, a native landrace of Saudi Arabia, tolerates local drought and high-salinity conditions and produces grain with diverse health-promoting phytochemicals. However, Hassawi has a long growth cycle, high cultivation costs, low productivity, and susceptibility to lodging. Here, to improve these undesirable traits via genome editing, we established efficient regeneration and Agrobacterium-mediated transformation protocols for Hassawi. In addition, we generated the first high-quality reference genome and targeted the key flowering repressor gene, Hd4, thus shortening the plant's lifecycle and height. Using CRISPR/Cas9 multiplexing, we simultaneously disrupted negative regulators of flowering time (Hd2, Hd4, and Hd5), grain size (GS3), grain number (GN1a), and plant height (Sd1). The resulting homozygous mutant lines flowered extremely early (∼56 days) and had shorter stems (approximately 107 cm), longer grains (by 5.1%), and more grains per plant (by 50.2%), thereby enhancing overall productivity. Furthermore, the awns of grains were 86.4% shorter compared to unedited plants. Moreover, the modified rice grain displayed improved nutritional attributes. As a result, the modified Hassawi rice combines several desirable traits that can incentivize large-scale cultivation and reduce malnutrition.

Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks.

Programmable site-specific nucleases (SSNs) hold extraordinary promise to unlock myriad gene editing applications in medicine and agriculture. However, developing small and specific SSNs is needed to overcome the delivery and specificity translational challenges of current genome engineering technologies. Structure-guided nucleases have been harnessed to generate double-strand DNA breaks but with limited success and translational potential. Here, we harnessed the power of peptide nucleic acids (PNAs) for site-specific DNA invasion and the generation of localized DNA structures that are recognized and cleaved by the eukaryotic resolvase AtMOC1 from Arabidopsis thaliana. We named this technology PNA-assisted Resolvase-mediated (PNR) editing. We tested the PNR editing concept in vitro and demonstrated its precise target specificity, examined the nucleotide requirement around the PNA invasion for the AtMOC1-mediated cleavage, mapped the AtMOC1-mediated cleavage sites, tested the role of different types and lengths of PNA molecules invasion into dsDNA for the AtMOC1-mediated cleavage, optimized the in vitro PNA invasion and AtMOC1 cleavage conditions such as temperature, buffer conditions, and cleavage time points, and demonstrated the multiplex cleavage for precise fragment release. We discuss the best design parameters for efficient, site-specific in vitro cleavage using PNR editors.

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology.

CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.

The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a CRISPR-Cas multiplexed diagnostic assay (CRISPRD) to detect cervical cancer-causing hrHPVs in one reaction (one-pot assay). We harnessed the compatibility of thermostable AapCas12b, TccCas13a, and HheCas13a nucleases with isothermal amplification and successfully detected HPV16 and HPV18, along with an internal control in a single-pot assay with a limit of detection of 10 copies and 100% specificity. This platform offers a rapid and practical solution for the multiplex detection of hrHPVs, which may facilitate large-scale hrHPV point-of-care screening. Furthermore, the CRISPRD platform programmability enables it to be adapted for the multiplex detection of any two nucleic acid biomarkers as well as internal control.

SCR106 splicing factor modulates abiotic stress responses by maintaining RNA splicing in rice.

Plants employ sophisticated molecular machinery to fine-tune their responses to growth, developmental, and stress cues. Gene expression influences plant cellular responses through regulatory processes such as transcription and splicing. Pre-mRNA is alternatively spliced to increase the genome coding potential and further regulate expression. Serine/arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Several studies have reported SR protein genes in the rice genome, subdivided into six subfamilies based on their domain structures. Here, we identified a new splicing factor in rice with an RNA recognition motif (RRM) and SR-dipeptides, which is related to the SR proteins, subfamily SC. OsSCR106 regulates pre-mRNA splicing under abiotic stress conditions. It localizes to the nuclear speckles, a major site for pre-mRNA splicing in the cell. The loss-of-function scr106 mutant is hypersensitive to salt, abscisic acid, and low-temperature stress, and harbors a developmental abnormality indicated by the shorter length of the shoot and root. The hypersensitivity to stress phenotype was rescued by complementation using OsSCR106 fused behind its endogenous promoter. Global gene expression and genome-wide splicing analysis in wild-type and scr106 seedlings revealed that OsSCR106 regulates its targets, presumably through regulating the alternative 3'-splice site. Under salt stress conditions, we identified multiple splice isoforms regulated by OsSCR106. Collectively, our results suggest that OsSCR106 is an important splicing factor that plays a crucial role in accurate pre-mRNA splicing and regulates abiotic stress responses in plants.

PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs.

Monitoring diseases caused by pathogens or by mutations in DNA sequences requires accurate, rapid, and sensitive tools to detect specific nucleic acid sequences. Here, we describe a new peptide nucleic acid (PNA)-based nucleic acid detection toolkit, termed PNA-powered diagnostics (PNA-Pdx). PNA-Pdx employs PNA probes that bind specifically to a target and are then detected in lateral flow assays. This can precisely detect a specific pathogen or genotype genomic sequence. PNA probes can also be designed to invade double-stranded DNAs (dsDNAs) to produce single-stranded DNAs for precise CRISPR-Cas12b-based detection of genomic SNPs without requiring the protospacer-adjacent motif (PAM), as Cas12b requires PAM sequences only for dsDNA targets. PNA-Pdx identified target nucleic acid sequences at concentrations as low as 2 copies/µL and precisely detected the SARS-CoV-2 genome in clinical samples in 40 min. Furthermore, the specific dsDNA invasion by the PNA coupled with CRISPR-Cas12b precisely detected genomic SNPs without PAM restriction. Overall, PNA-Pdx provides a novel toolkit for nucleic acid and SNP detection as well as highlights the benefits of engineering PNA probes for detecting nucleic acids.

Programmable site-specific DNA double-strand breaks via PNA-assisted prokaryotic Argonautes.

Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.

Impacts of dietary zinc oxide nanoparticles on the growth and immunity of Nile tilapia could be ameliorated using Nigella sativa oil.

BACKGROUND: Zinc nanoparticles are documented to be harmful to fish because their accumulation in fish bring about many irreversible changes in their health. Nigella sativa and its oil have been endorsed in aquaculture to improve fish health. METHODS: Two hundred seventy experimental fish (113 ± 5 g body weight) were divided into 6 groups G1-6; control fish fed a diet without any treatment (G1), 0.3% of NSO (G2), 0.5% of NSO (G3), ZnO NPs (40 mg/kg diet) (G4), 0.3% of NSO and ZnO NPs (40 mg/kg diet) (G5), 0.5% of NSO and ZnO NPs (40 mg/kg diet) (G6), the trial lasted for six weeks. RESULTS: Growth performance was enhanced in fish received diets containing NSO, final weight (FW), weight gain (WG), daily weight gain (DWG), and relative growth rate (RGR) were significantly increased with lower food conversion ratios (FCR) compared to the control. The hepatic glutathione peroxidase (GPx), catalase (CAT), and metallothionein (MT) were increased in response to ZnO NPs stress and only 0.5% NSO supplementation could ameliorate such increment. The immune-related genes [interleukin1-beta (IL-1ß), tumor necrosis factor-beta (TNF-ß), transforming growth factor-beta 2 (TGF-ß2) and C-type lysozyme] as well as growth-related gene [insulin-like growth factor 1 (IGF1)] in liver showed an upregulation in fish fed with NSO diets. Administration of ZnO NPs lowered the resistance of Oreochromis niloticus against bacterial infection with Aeromonas hydrophila and NSO could enhance the immunity in the highest tested concentration (0.5%) (G6). CONCLUSIONS: The obtained results implied that NSO could enhance the oxidative and immune status of O. niloticus which could compensate ZnO NPs stress as well as experimental infection of a virulent strain of A. hydrophila. Our results revealed that NSO might increase fish growth and immunity only at a high dose (0.5%).

Potential of curcumin and niacin-loaded targeted chitosan coated liposomes to activate autophagy in hepatocellular carcinoma cells: An in vitro evaluation in HePG2 cell line.

The objective of this study is to activate autophagy in hepatocellular carcinoma for the enhancement of its cellular degradation. Liposomes incorporated chitosan in the core used to improve the stability of lecithin and increase the niacin loading efficiency. Additionally, curcumin as a hydrophobic molecule entrapped into liposomal layers and used as a face layer to minimize the release of niacin in physiological pH 7.4. Folic acid-conjugated chitosan was used to facilitate the delivery of liposomes into a specific location of cancer cells. TEM, UV Visible spectrophotometer, and FTIR confirmed the successful liposomal formation and good encapsulation efficiency. Based on the cellular proliferation of HePG2, the results revealed that there was a significant inhibition of growth rate of HePG2 after 48 h of incubation at a concentration of 100 µg/mL by 91 % ± 1 %, P ≤ 0.002 (pure niacin), 55 % ± 3 %, P ≤ 0.001 (pure curcumin), 83 % ± 1.5 %, P ≤ 0.001 (niacin NPs), and 51 % ± 1.5 % P ≤ 0.0001 (curcumin-niacin NPs) of relative to the control. Increasingly, The expression of mRNA of mTOR was significantly increased by 0.72 ± 0.08 P ≤ 0.001, 1 ± 0.1, 0. P ≤ 0.001, 5 ± 0.07 P ≤ 0.01, and 1.3 ± 0.02 P ≤ 0.001 folds) in pure niacin, pure curcumin, niacin NPs and curcumin -niacin NPs, respectively, relative to the control with an expression of 0.3 ± 0.08. Additionally, the expression of p62 mRNA was significantly increased by 0.92 ± 0.07 P ≤ 0.05, 1.7 ± 0.07 P ≤ 0.0001, 0.72 ± 0.08 P ≤ 0.5, and 2.1 ± 0.1 P ≤ 0.0001 folds relative to that of the control with an expression of 0.72 ± 0.08. The results highlight the efficient therapies of biomaterials derived from natural sources that can be used in cancer therapies instead of traditional chemotherapies.

Multi-omics resources for targeted agronomic improvement of pigmented rice.

Pigmented rice (Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies of 5 pigmented rice varieties and evaluated the genetic variation among 51 pigmented rice varieties by resequencing an additional 46 varieties. Phylogenetic analyses divided the pigmented varieties into four varietal groups: Geng-japonica, Xian-indica, circum-Aus and circum-Basmati. Metabolomics and ionomics profiling revealed that black rice varieties are rich in aromatic secondary metabolites. We established a regeneration and transformation system and used CRISPR-Cas9 to knock out three flowering time repressors (Hd2, Hd4 and Hd5) in the black Indonesian rice Cempo Ireng, resulting in an early maturing variety with shorter stature. Our study thus provides a multi-omics resource for understanding and improving Asian pigmented rice.

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline.

Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2 µM in a short time, requiring only 15 min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.

Simultaneous Administration of Bevacizumab with Bee-Pollen Extract-Loaded Hybrid Protein Hydrogel NPs Is a Promising Targeted Strategy against Cancer Cells.

Bevacizumab (Bev) a humanized monoclonal antibody that fights vascular endothelial growth factor A (VEGF-A). It was the first specifically considered angiogenesis inhibitor and it has now become the normative first-line therapy for advanced non-small-cell lung cancer (NSCLC). In the current study, polyphenolic compounds were isolated from bee pollen (PCIBP) and encapsulated (EPCIBP) inside moieties of hybrid peptide-protein hydrogel nanoparticles in which bovine serum albumin (BSA) was combined with protamine-free sulfate and targeted with folic acid (FA). The apoptotic effects of PCIBP and its encapsulation (EPCIBP) were further investigated using A549 and MCF-7 cell lines, providing significant upregulation of Bax and caspase 3 genes and downregulation of Bcl2, HRAS, and MAPK as well. This effect was synergistically improved in combination with Bev. Our findings may contribute to the use of EPCIBP simultaneously with chemotherapy to strengthen the effectiveness and minimize the required dose.

HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions.

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts.