Ligand-free biodegradable poly(beta-amino ester) nanoparticles for targeted systemic delivery of mRNA to the lungs.

Non-viral nanoparticles (NPs) have seen heightened interest as a delivery method for a variety of clinically relevant nucleic acid cargoes in recent years. While much of the focus has been on lipid NPs, non-lipid NPs, including polymeric NPs, have the possibility of improved efficacy, safety, and targeting, especially to non-liver organs following systemic administration. A safe and effective systemic approach for intracellular delivery to the lungs could overcome limitations to intratracheal/intranasal delivery of NPs and improve clinical benefit for a range of diseases including cystic fibrosis. Here, engineered biodegradable poly (beta-amino ester) (PBAE) NPs are shown to facilitate efficient delivery of mRNA to primary human airway epithelial cells from both healthy donors and individuals with cystic fibrosis. Optimized NP formulations made with differentially endcapped PBAEs and systemically administered in vivo lead to high expression of mRNA within the lungs in BALB/c and C57 B/L mice without requiring a complex targeting ligand. High levels of mRNA-based gene editing were achieved in an Ai9 mouse model across bronchial, epithelial, and endothelial cell populations. No toxicity was observed either acutely or over time, including after multiple systemic administrations of the NPs. The non-lipid biodegradable PBAE NPs demonstrate high levels of transfection in both primary human airway epithelial cells and in vivo editing of lung cell types that are targets for numerous life-limiting diseases particularly single gene disorders such as cystic fibrosis and surfactant deficiencies.

Comparing the benthic nitrogenase activity and diazotrophic community assembly of three large river-connected freshwater lakes in eastern China.

Biological nitrogen fixation (BNF) is a crucial process that provides bioavailable nitrogen and supports primary production in freshwater lake ecosystems. However, the characteristics of diazotrophic community and nitrogenase activity in freshwater lake sediments remain poorly understood. Here, we investigated the diazotrophic communities and nitrogenase activities in the sediments of three large river-connected freshwater lakes in eastern China using 15N-isotope tracing and nifH sequencing. The sediments in these lakes contained diverse nitrogenase genes that were phylogenetically grouped into Clusters I and III. The diazotrophic communities in the sediments were dominated by stochastic processes in Hongze Lake and Taihu Lake, which had heterogeneous habitats and shallower water depths, while in Poyang Lake, which had deeper water and a shorter hydraulic retention time, the assembly of the diazotrophic community in the sediments was dominated by homogeneous selection processes. Temperature and water depth were also found the key environmental factors affecting the sediment diazotrophic communities. Sediment nitrogenase activities varied in the three lakes and within distinct regions of an individual lake, ranging from 0 to 14.58 nmol/(kg·hr). Nitrogenase activity was significantly correlated with ferric iron, total phosphorus, and organic matter contents. Our results suggested that freshwater lake sediment contain high diversity of nitrogen-fixing microorganisms with potential metabolic diversity, and the community assembly patterns and nitrogenase activities varied with the lake habitat.

Novel strategies in HPV­16­related cervical cancer treatment: An in vitro study of combined siRNA-E5 with oxaliplatin and ifosfamide chemotherapy.

BACKGROUND: Cervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. METHODS: The CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. RESULTS: E5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10-8 M (45.36 µg/ml) to 6.71 × 10-8 M (26.66 µg/ml) and Ifosfamide from 12.52 × 10-5 M (32.7 µg/ml) to 8.206 × 10-5 M (21.43 µg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. CONCLUSION: The study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.

Construction of Small Genes with Repetitive Elements Using Oligo Extension and Golden Gate Assembly.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences and results in synthesis failure or delays by DNA synthesis vendors. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible overhangs. Then synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate assembly.

Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria.

Bacterial small RNAs (sRNAs) are well known for their ability to modulate gene expression at the post-transcriptional level. Their rather simple and modular organization provides the user with defined building blocks for synthetic biology approaches. In this chapter, we introduce a plasmid series for Escherichia coli and describe protocols for fast and efficient construction of synthetic sRNA expression plasmids based on Golden Gate assembly. In addition, we present the G-GArden tool, which assists with the design of oligodeoxynucleotides and overhangs for scarless assembly strategies. We propose that the presented procedures are suitable for many applications in different bacteria, which are related to E. coli and beyond.

Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.

Evaluation of Gene Expression Profiling by QuantiGene™ 2.0 RNA Assay.

QuantiGene™ 2.0 technique could be used to investigate the gene expression signature of the immune system senescence and thus to understand the molecular mechanism involved in the defects of the immune response during aging.QuantiGene™ 2.0 technique is a multiplex platform allowing the simultaneous analysis of several target RNA molecules (up to 80) present in a single sample. QuantiGene Assays use an accurate method for multiplexed or for single gene expression quantitation. QuantiGene 2.0 uses magnetic beads which are dyed internally with two fluorescence dyes, exhibiting a unique spectral signal and providing specificity and multiplexing capability of the technique. QuantiGene Assays incorporate branched-DNA technology for gene expression profiling.Branched-DNA system is responsible for the high sensitivity of the system. In fact, it permits to detect low levels of mRNA molecules. This branched-DNA system allows for the direct measurement of RNA transcripts by using signal amplification rather than target amplification. The assay protocol is spread over 2 days. First, immune cells are lysed to release the target RNA, which is incubated with oligonucleotide probe set targeted with beads capable to hybridize with the target RNA. Signal amplification is performed by sequential hybridization of the branched-DNA pre-amplifier, amplifier, and label probe molecules. The last step involves the incubation with Streptavidin-conjugated R-phycoerythrin. The fluorescent reporter generates a signal directly proportional to the levels of RNA molecules present in the cells. Luminex instrument evaluates the median intensity of fluorescence, which is proportional to the number of RNA target molecules present in the cells.

Analysis of Cytokine-Inducible SH2-Containing Protein (CISH) in the Study of Immunosenescence.

The aging immune system undergoes significant changes, leading to a state known as immunosenescence. Understanding the molecular mechanisms underlying immunosenescence is crucial for developing targeted interventions to enhance immune functions in older individuals. This bio-protocol review focuses on the application of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the mRNA quantification of cytokine-inducible SH2-containing protein (CISH), an immune regulator overexpressed in T-cell responses from older adults. We outline a comprehensive protocol for the quantitative assessment of CISH mRNA expression, providing a valuable tool for researchers investigating immunosenescence.

From the cauldron of conflict: Endogenous gene regulation by piRNA and other modes of adaptation enabled by selfish transposable elements.

Transposable elements (TEs) provide a prime example of genetic conflict because they can proliferate in genomes and populations even if they harm the host. However, numerous studies have shown that TEs, though typically harmful, can also provide fuel for adaptation. This is because they code functional sequences that can be useful for the host in which they reside. In this review, I summarize the "how" and "why" of adaptation enabled by the genetic conflict between TEs and hosts. In addition, focusing on mechanisms of TE control by small piwi-interacting RNAs (piRNAs), I highlight an indirect form of adaptation enabled by conflict. In this case, mechanisms of host defense that regulate TEs have been redeployed for endogenous gene regulation. I propose that the genetic conflict released by meiosis in early eukaryotes may have been important because, among other reasons, it spurred evolutionary innovation on multiple interwoven trajectories - on the part of hosts and also embedded genetic parasites. This form of evolution may function as a complexity generating engine that was a critical player in eukaryotic evolution.

CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

Arsenic methylation and microbial communities in paddy soils under alternating anoxic and oxic conditions.

Arsenic (As) methylation in soils affects the environmental behavior of As, excessive accumulation of dimethylarsenate (DMA) in rice plants leads to straighthead disease and a serious drop in crop yield. Understanding the mobility and transformation of methylated arsenic in redox-changing paddy fields is crucial for food security. Here, soils including un-arsenic contaminated (N-As), low-arsenic (L-As), medium-arsenic (M-As), and high-arsenic (H-As) soils were incubated under continuous anoxic, continuous oxic, and consecutive anoxic/oxic treatments respectively, to profile arsenic methylating process and microbial species involved in the As cycle. Under anoxic-oxic (A-O) treatment, methylated arsenic was significantly increased once oxygen was introduced into the incubation system. The methylated arsenic concentrations were up to 2-24 times higher than those in anoxic (A), oxic (O), and oxic-anoxic (O-A) treatments, under which arsenic was methylated slightly and then decreased in all four As concentration soils. In fact, the most plentiful arsenite S-adenosylmethionine methyltransferase genes (arsM) contributed to the increase in As methylation. Proteobacteria (40.8%-62.4%), Firmicutes (3.5%-15.7%), and Desulfobacterota (5.3%-13.3%) were the major microorganisms related to this process. These microbial increased markedly and played more important roles after oxygen was introduced, indicating that they were potential keystone microbial groups for As methylation in the alternating anoxic (flooding) and oxic (drainage) environment. The novel findings provided new insights into the reoxidation-driven arsenic methylation processes and the model could be used for further risk estimation in periodically flooded paddy fields.

MiR-6721-5p as a natural regulator of Meta-VCL is upregulated in the serum of patients with coronary artery disease.

Background: Coronary artery disease (CAD), the leading cause of mortality globally, arises from atherosclerotic blockage of the coronary arteries. Meta-vinculin (meta-VCL), a large spliced isoform of VCL, co-localizes in muscular adhesive structures and plays significant roles in cardiac physiology and pathophysiology. This study aimed to identify microRNAs (miRNAs) regulating meta-VCL expression and investigate the expression alterations of the miRNAs of interest and meta-VCL as potential biomarkers in the serum of CAD patients. Methods: Bioinformatics tools were employed to select miRNAs targeting meta-VCL. Cell-based ectopic expression analysis and a dual-luciferase assay were used to examine the interactions between miRNAs and meta-VCL. An ELISA assessed the concentrations of interleukin-6 (IL-6), IL-10, and tumor necrosis factor-α (TNF-α). MiRNA and meta-VCL expression patterns and biomarker suitability were evaluated in serum samples from CAD and non-CAD individuals using real-time PCR. A cardiac cell-line data set and CAD blood exosome samples were analyzed using bioinformatics and ROC curve analyses, respectively. Results: miR-6721-5p directly interacted with the putative target sites at the 3'-UTR of meta-VCL and regulated its expression. IL-10 and TNF-α concentrations, which may act as anti-inflammatory factors, decreased following miR-6721-5p upregulation and meta-VCL downregulation. Bioinformatics and experimental expression analyses confirmed downregulated meta-VCL expression and upregulated miR-6721-5p expression in CAD samples. ROC curve analysis yielded an AUC score of 0.705 (P = 0.018), indicating the potential suitability of miR-6721-5p as a biomarker for CAD. Conclusions: miR-6721-5p plays a regulatory role in meta-VCL expression and may contribute to CAD development by reducing anti-inflammatory factors. These findings suggest that miR-6721-5p could serve as a novel biomarker in the pathogenesis of CAD.

Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout.

Bacillus licheniformis is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple genes. To address this limitation, the CRISPR-Cpf1 system has been developed, enabling multiplexed gene editing across various microorganisms. Key to the efficient gene editing capability of this system is the rigorous screening of highly effective expression elements to achieve conditional expression of protein Cpf1. In this study, we employed mCherry as a reporter gene and harnessed P mal for regulating the expression of Cpf1 to establish the CRISPR-Cpf1 gene editing system in Bacillus licheniformis. Our system achieved a 100 % knockout efficiency for the single gene vpr and up to 80 % for simultaneous knockout of the double genes epr and mpr. Furthermore, the culture of a series of protease-deficient strains revealed that the protease encoded by aprE contributed significantly to extracellular enzyme activity (approximately 80 %), whereas proteases encoded by vpr, epr, and mpr genes contributed to a smaller proportion of extracellular enzyme activity. These findings provide support for effective molecular modification and metabolic regulation in industrial organisms.

"All in one" lipid-polymer nanodelivery system for gene therapy of ischemic diseases.

Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.

Prediction of Enhancer-Gene Interactions Using Chromatin-Conformation Capture and Epigenome Data Using STARE.

Disentangling the relationship of enhancers and genes is an ongoing challenge in epigenomics. We present STARE, our software to quantify the strength of enhancer-gene interactions based on enhancer activity and chromatin contact data. It implements the generalized Activity-by-Contact (gABC) score, which allows predicting putative target genes of candidate enhancers over any desired genomic distance. The only requirement for its application is a measurement of enhancer activity. In addition to regulatory interactions, STARE calculates transcription factor (TF) affinities on gene level. We illustrate its usage on a public single-cell data set of the human heart by predicting regulatory interactions on cell type level, by giving examples on how to integrate them with other data modalities, and by constructing TF affinity matrices.

Learning Enhancer-Gene associations from Bulk Transcriptomic and Epigenetic Sequencing Data with STITCHIT.

To reveal gene regulation mechanisms, it is essential to understand the role of regulatory elements, which are possibly distant from gene promoters. Integrative analysis of epigenetic and transcriptomic data can be used to gain insights into gene-expression regulation in specific phenotypes. Here, we discuss STITCHIT, an approach to dissect epigenetic variation in a gene-specific manner across many samples for the identification of regulatory elements without relying on peak calling algorithms. The obtained genomic regions are then further refined using a regularized linear model approach, which can also be used to predict gene expression. We illustrate the use of STITCHIT using H3k27ac ChIP-seq and RNA-seq data from the International Human Epigenome Consortium (IHEC).

Comparing Gene Expression Between Planktonic and Biofilm Cells of Foodborne Bacterial Pathogens Through RT-qPCR.

Like most microorganisms, important foodborne pathogenic bacteria, such as Salmonella enterica, Listeria monocytogenes, and several others as well, can attach to surfaces, of either abiotic or biotic nature, and create biofilms on them, provided the existence of supportive environmental conditions (e.g., permissive growth temperature, adequate humidity, and nutrient presence). Inside those sessile communities, the enclosed bacteria typically present a gene expression profile that differs from the one that would be displayed by the same cells growing planktonically in liquid media (free-swimming cells). This altered gene expression has important consequences on cellular physiology and behavior, including stress tolerance and induction of virulence. In this chapter, the methodology to use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to monitor and comparatively quantify expression changes in preselected genes of bacteria between planktonic and biofilm growth modes is presented.

Illumina MiSeq 16S rRNA Gene Library Preparation for Poultry Processing Microbiome Analyses.

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety.This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

Systemic messenger RNA replacement therapy is effective in a novel clinically relevant model of acute intermittent porphyria developed in non-human primates.

OBJECTIVE: Acute intermittent porphyria (AIP) is a rare metabolic disorder caused by haploinsufficiency of hepatic porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthesis. Individuals with AIP experience neurovisceral attacks closely associated with hepatic overproduction of potentially neurotoxic heme precursors. DESIGN: We replicated AIP in non-human primates (NHPs) through selective knockdown of the hepatic PBGD gene and evaluated the safety and therapeutic efficacy of human PBGD (hPBGD) mRNA rescue. RESULTS: Intrahepatic administration of a recombinant adeno-associated viral vector containing short hairpin RNA against endogenous PBGD mRNA resulted in sustained PBGD activity inhibition in liver tissue for up to 7 months postinjection. The administration of porphyrinogenic drugs to NHPs induced hepatic heme synthesis, elevated urinary porphyrin precursors and reproduced acute attack symptoms in patients with AIP, including pain, motor disturbances and increased brain GABAergic activity. The model also recapitulated functional anomalies associated with AIP, such as reduced brain perfusion and cerebral glucose uptake, disturbances in hepatic TCA cycle, one-carbon metabolism, drug biotransformation, lipidomic profile and abnormal mitochondrial respiratory chain activity. Additionally, repeated systemic administrations of hPBGD mRNA in this AIP NHP model restored hepatic PBGD levels and activity, providing successful protection against acute attacks, metabolic changes in the liver and CNS disturbances. This approach demonstrated better efficacy than the current standards of care for AIP. CONCLUSION: This novel model significantly expands our understanding of AIP at the molecular, biochemical and clinical levels and confirms the safety and translatability of multiple systemic administration of hPBGD mRNA as a potential aetiological AIP treatment.