Reverse Transcription-polymerase Chain Reaction Analysis Research Articles

Development of cartilage tissue using a stirred bioreactor and human iPSC-derived limb bud mesenchymal cells

Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.

Polyamine signaling communications play a key role in regulating the pathogenicity of Dickeya fangzhongdai.

Dickeya fangzhongdai is a devastating bacterial pathogen infecting a wide range of crops and ornamental plants worldwide. As a newly identified bacterial species in 2016, the regulatory mechanisms that govern its virulence are still a mystery. In this study, we explored the potential roles of polyamine-mediated cell-to-cell communication in regulation of D. fangzhongdai virulence. Null mutation of speA and speC in D. fangzhongdai strain ZXC1, which encodes polyamine biosynthesis through arginine and ornithine pathways, respectively, dramatically reduced bacterial motility, decreased production of plant cell wall degradation (PCWD) enzymes, and attenuated the bacterial virulence on taro and potato. We then tested the effect of various polyamine molecules in the restoration of the mutant phenotypes and showed that putrescine was the most potent signal in the regulation of virulence traits in strain ZXC1. In addition, we found that taro extract contained active signals to rescue putrescine-deficient phenotypes. High-performance liquid chromatography mass spectrometry analysis validated the speA was essential for production of putrescine in D. fangzhongdai ZXC1. We further showed that the putrescine transporters PotF and PlaP are required for putrescine-mediated cell-to-cell communication and virulence against taro and potato tubers. quantitative reverse transcription-PCR analysis demonstrated that putrescine influences the pathogenicity of D. fangzhongdai ZXC1 by regulating the expression of PCWD enzymes, bacterial chemotaxis, and flagellar-related genes. The findings from this study shed a new light for elucidating the pathogenic mechanisms of D. fangzhongdai and present useful clues for developing relevant disease control strategies. IMPORTANCE Dickeya fangzhongdai is a newly identified plant bacterial pathogen with a wide host range. A clear understanding of the cell-to-cell communication systems that modulate the bacterial virulence is of key importance for elucidating its pathogenic mechanisms and for disease control. In this study, we present evidence that putrescine molecules from the pathogen and host plants play an essential role in regulating the bacterial virulence. The significance of this study is in (i) demonstrating that putrescine signaling system regulates D. fangzhongdai virulence mainly through modulating the bacterial motility and production of PCWD enzymes, (ii) outlining the signaling and regulatory mechanisms with which putrescine signaling system modulates the above virulence traits, and (iii) validating that D. fangzhongdai could use both arginine and ornithine pathways to synthesize putrescine signals. To our knowledge, this is the first report to show that putrescine signaling system plays a key role in modulating the pathogenicity of D. fangzhongdai.

Ameliorating effect of nanoparticles and seeds’ heat pre-treatment on soybean plants exposed to sea water salinity

Impairing plant growth and reducing crop production, salinity is considered as major problem in modern agriculture. The current study aimed to investigate the role of seeds' heat pretreatment at 45 °C as well as application of two different nanoparticles nanosilica (N1) and nanoselenium (N2) in reducing salinity stress in three genotypes of Egyptian commercial soybeans (Glycine max L.). Two levels of salt stress using diluted sea water (1/12 and 1/6) were tested either alone or in combination with protective treatments. Obtained results revealed that salinity caused a significant reduction in all tested physiological parameters such as germination rate and membrane stability in soybean plants. A significant reduction in mitotic index and arrest in metaphase were recorded under both tested levels of salinity. It was also revealed that chromosomal abnormalities in soybean plants were positively correlated with the applied salinity concentrations. The fragmentation effect of salinity on the nuclear DNA was investigated and confirmed using Comet assay analysis. Seeds heat pre-treatment (45 °C) and both types of nanoparticles' treatments yielded positive effects on both the salt-stressed and unstressed plants. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis for salt stress responsive marker genes revealed that most studied genes (CAT, APX, DHN2, CAB3, GMPIPL6 and GMSALT3) responded favorably to protective treatments. The modulation in gene expression pattern was associated with improving growth vigor and salinity tolerance in soybean plants. Our results suggest that seeds’ heat pretreatment and nanoparticle applications support the recovery against oxidative stresses and represent a promising strategy for alleviating salt stress in soybean genotypes.

Ultrasound-triggered release of miR-199a-3p from liposome nanobubbles for enhanced hepatocellular carcinoma treatment

This study was aimed to develop an efficient tumour-targeted liposome nanobubbles (LNBs) system using ultrasound-targeted nanobubble destruction for enhanced release and transfection of miRNA-199a-3p in hepatocellular carcinoma (HCC) therapy. The prepared LNBs comprised a polyethylene glycol-modified liposome shell and a perfluoropentane (PFP) core. MiRNA-199a-3p was attached to the nanocomposite surface via electrostatic adsorption, while RGD peptide functionalized the LNBs surface for enhanced HCC cell targeting, namely PFP@miR-RGD-LNBs. The LNBs were spherical with a narrow size distribution. The gene-loaded LNBs effectively condensed miR-199a-3p and protected it from enzymatic degradation. Low-intensity focused ultrasound (LIFU) promoted a fast release of miR-199a-3p from the prepared LNBs, thereby enhancing therapeutic effects. The combined application of PFP@miR-RGD-LNBs and LIFU exhibited a more potent inhibitory effect on HepG2 cells than the other groups, potentially due to LIFU promoting rapid and efficient gene release at the target site and increasing cell membrane permeability. Quantitative reverse transcription-polymerase chain reaction analysis revealed significantly increased mRNA expression levels of key apoptosis markers (Bad, Bax, Caspase-9 and Caspase-3) in the PFP@miR-RGD-LNBs + LIFU group compared to other groups. These findings suggest that the prepared LNBs are highly likely to be promising candidates for further exploration of HCC gene delivery and therapy.

Esculetin improves murine mastitis induced by streptococcus isolated from bovine mammary glands by inhibiting NF-κB and MAPK signaling pathways

Cow mastitis, caused by Streptococcus infection of the mammary glands, is a common clinical disease that can lead to decreased milk quality and threaten animal welfare and performance. Esculetin (ESC) is a coumarin with anti-inflammatory and anti-asthmatic effects. However, whether ESC has therapeutic effects on mastitis remains unexplored. This study was conducted to investigate the protective effect of ESC against murine mastitis caused by Streptococcus isolated from bovine mammary glands and elucidate the underlying mechanisms. Streptococcus uberis was used to construct a mouse model of mastitis. The results showed that the mice exhibited edema and thickening of the acinar wall with inflammatory infiltration after S. uberis treatment. Intraperitoneal injection of ESC significantly reduced inflammatory cell infiltration, restored normal physiological function, and inhibited the production of the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that ESC reduced P38 phosphorylation, further inhibited the influence of mammary Streptococcus on cytoplasmic translocation of nuclear factor-κB (P65), and inhibited the transcriptional activation of P65, thus inhibiting the generation of inflammatory cells. Collectively, ESC may inhibit mitogen-activated protein kinase and nuclear factor-κB, thereby highlighting its potential for the treatment and prevention of mastitis.

WNT4 promotes macrophage polarization via granulosa cell M-CSF and reduces granulosa cell apoptosis in endometriosis

BackgroundWNT4 gene polymorphism are common in endometriosis and may functionally link estrogen and estrogen receptor signaling. Previous study confirmed estrogen and estrogen receptor signaling recruit macrophage to promote the pathogenesis of endometriosis. To investigate the effect of WNT4 in endometriosis involved in macrophage polarization and whether WNT4 could reduce the apoptosis of granulosa cells. MethodsAn observational study consisting of 8 cases of women with endometriosis (diagnosed by surgery and histology) and 22 mice of endometriosis animal model was conducted. Granulosa cells were isolated from 16 patients with endometriosis and co-cultured with macrophage under WNT4 treatment using TUNEL assay, quantitative reverse transcription PCR, flow cytometry and ELISA analysis. 22 mice of endometriosis animal model confirmed the WNT4 treatment effects using histology and immunohistochemistry, Western blot and flow cytometry. ResultsWe observed that the apoptotic proportion of granulosa cells was significantly decreased and M2 macrophage was significantly increased after WNT4 treatment during the granulosa cell and macrophage co-culture system. To reveal the underlying mechanism for this, we conducted a series of experiments and found that high expression of granulosa cell M-CSF led to the M2 polarization of macrophages. The animal model also suggested that the anti-apoptotic effect of WNT4 on granulosa cells were conducted by the M2 polarized macrophage. ConclusionsWNT4 could reduce granulosa cell apoptosis and improve ovarian reserve by promoting macrophage polarization in endometriosis. M-CSF secreted by granulosa cell after WNT4 treatment was the main mediator of macrophage polarization.

Rapid Environmental Monitoring, Capture, and Destruction Activities of SARS-CoV-2 and Bacterial Pathogens During the COVID-19 Health Emergency.

The SARS-CoV-2 pandemic is a health emergency for occupational healthcare workers at COVID-19 hospital wards in Italy. The objective of the study was to investigate the bioreactor's effectivity in monitoring and improving air quality via detection, capture, and destruction of the SARS-CoV-2 virus and bacterial pathogens, reducing the risk of transmission among healthcare workers. Bioreactors are a demonstrated effective biomonitoring system. We implemented a methodological approach wherein they were placed at various hospitals treating COVID-19 patients in Italy. The detection of the SARS-CoV-2 virus was achieved through rapid biomonitoring testing of the solutes from the AIRcel bioreactors via SARS-CoV-2 rapid test antigen and consecutive reverse transcription-polymerase chain reaction (RT-PCR) analysis with the multiplex platform (XABT) and the real-time PCR rotor-gene. The marked presence of the SARS-CoV-2 virus was found in multiple water samples via the detection of ORF1ab + N and/or E gene involved in gene expression and cellular signaling of the SARS-CoV-2 virus. The AIRcel bioreactors were able to neutralize the virus and bacterial pathogens effectively as traces of the viruses and bacteria were no longer found in multiple solute samples after an overnight period. Transmission of COVID-19 via bioaerosols, transmitted by infected patients, remains a viable threat for health workers. AIRcel bioreactors allow for rapid biomonitoring testing for early virus detection within the environment, reducing the risk of exponential contagion exposure and maintaining good air quality without endangering health workers. This sameprotocol can also be extended to public spaces as a bio-monitoring hotspot tool for early detection.

Differentially expressed circRNAs in peripheral blood samples as potential biomarkers and therapeutic targets for acute angle-closure glaucoma

Glaucoma is the leading cause of irreversible blindness globally. Circular RNAs (circRNAs) play vital roles in various biological processes as microRNA (miRNA) sponges and, thus, have been investigated as potential biomarkers and therapeutic targets in numerous human diseases. However, the underlying mechanisms of circRNAs in the pathogenesis of glaucoma remain unclear. Therefore, transcriptome sequencing was performed to identify relevant circRNAs in peripheral blood samples from patients with primary angle-closure glaucoma. Bioinformatics analysis was performed to investigate the potential roles of differentially expressed circRNAs (DEcircRNAs) in the pathogenesis of glaucoma. In total, 481 differentially expressed genes in addition to 345 DEcircRNAs were identified in patients with glaucoma. Based on a public database, targeted gene analysis identified 11 DEcircRNAs that potentially regulate the expression of five genes as miRNA sponges in glaucoma. In addition, quantitative reverse transcription PCR analysis verified that expression of the circRNA hsa-circ-0000745 was positively correlated with the expression of NEAT1 as a potential target gene. These results suggest that DEcircRNAs are involved in a gene expression regulatory network related to immune cell function and progression of glaucoma. Thus, DEcircRNAs in peripheral blood are potential biomarkers and therapeutic targets for glaucoma.

Genome-wide analysis of blueberry B-box family genes and identification of members activated by abiotic stress

BackgroundB-box (BBX) proteins play important roles in regulating plant growth, development, and abiotic stress responses. BBX family genes have been identified and functionally characterized in many plant species, but little is known about the BBX family in blueberry (Vaccinium corymbosum).ResultIn this study, we identified 23 VcBBX genes from the Genome Database for Vaccinium (GDV). These VcBBXs can be divided into five clades based on gene structures and conserved domains in their encoded proteins. The prediction of cis-acting elements in the upstream sequences of VcBBX genes and protein–protein interactions indicated that VcBBX proteins are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of transcriptome deep sequencing (RNA-seq) data showed that VcBBX genes exhibited organ-specific expression pattern and 11 VcBBX genes respond to ultraviolet B (UV-B) radiation. The co-expression analysis revealed that the encoded 11 VcBBX proteins act as bridges integrating UV-B and phytohormone signaling pathways in blueberry under UV-B radiation. Reverse-transcription quantitative PCR (RT-qPCR) analysis showed that most VcBBX genes respond to drought, salt, and cold stress. Among VcBBX proteins, VcBBX24 is highly expressed in all the organs, not only responds to abiotic stress, but it also interacts with proteins in UV-B and phytohormone signaling pathways, as revealed by computational analysis and co-expression analysis, and might be an important regulator integrating abiotic stress and phytohormone signaling networks.ConclusionsTwenty-three VcBBX genes were identified in blueberry, in which, 11 VcBBX genes respond to UV-B radiation, and act as bridges integrating UV-B and phytohormone signaling pathways according to RNA-seq data. The expression patterns under abiotic stress suggested that the functional roles of most VcBBX genes respose to drought, salt, and cold stress. Our study provides a useful reference for functional analysis of VcBBX genes and for improving abiotic stress tolerance in blueberry.

Molecular Identification and Expression Analysis of an Intelectin Gene in the Yellow Catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae)

Intelectins (ITLNs) are a family of calcium-dependent lectins with carbohydrate-binding capacity, are distributed across various vertebrates, and play an important role in the innate immune response against pathogen infection. The yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. The aim of this study was to quantify the gene expression of ITLN in response to pathogen-associated molecular patterns (PAMPs) stimulation. Here, the ITLN gene of P. fulvidraco was characterized and named PfITLN. The full-length cDNA of PfITLN was 1132 bp, including a 5’-untranslated region (UTR) of 140 bp, a 3’-UTR of 110 bp, and an open reading frame (ORF) of 882 bp encoding a polypeptide of 293 amino acids, which contains a signal peptide and two fibrinogen-related domains (FReDs). PfITLN had a molecular weight of 32.39 kDa with a theoretical pI of 5.03. The deduced PfITLN amino acid sequence had 81%, 64%, and 55% homology with Ictalurus furcatus, Danio rerio, and Homo sapiens, respectively. Moreover, the predicted tertiary protein structure of PfITLN was highly similar to that of other animals, and phylogenetic analysis showed that the PfITLN protein was close to those of other Teleostei. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed PfITLN expression in all examined tissues, with the highest abundance seen in the liver, followed by the head kidney, spleen, trunk kidney, and muscle. After PAMP infection with lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (poly I:C), the expression levels of PfITLN were significantly upregulated at different time points. These results suggested that PfITLN might be involved in innate immunity.

Curcumin's Dose-Dependent Attenuation of Gastric Cancer Cell Progression Via the PI3K Pathway Blockade.

Background: Gastric cancer stands as a primary cause of cancer-related deaths worldwide, making the discovery of new therapeutic agents essential for enhancing treatment outcomes. Curcumin, a polyphenolic compound found in turmeric (Curcuma longa), has demonstrated potential in multiple cancer types due to its anti-cancer characteristics. This research aimed to examine the impact of curcumin on gastric cancer cell growth, migration, and invasion, as well as its influence on the phosphoinositide 3-kinase (PI3K) signaling cascade. Methods: Gastric cancer cell lines were exposed to varying curcumin concentrations, followed by assessments of cell viability, migration, and invasion. Furthermore, gene and protein expression levels associated with the PI3K signaling cascade were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Results: The findings revealed a dose-dependent decrease in cell viability, migration, and invasion in gastric cancer cells treated with curcumin. Additionally, curcumin administration led to the downregulation of key genes and proteins within the PI3K signaling process, such as PI3K, Akt, and mTOR. Conclusion: These findings propose that curcumin may exercise its anti-cancer effects on gastric cancer cells, partly by suppressing the PI3K signaling pathway. This study's outcomes support curcumin's potential as a therapeutic agent for gastric cancer and encourage further exploration of its underlying molecular mechanisms and in vivo effectiveness.

Detection and molecular characterization of virulent Newcastle disease virus (subgenotype VII.2) in broiler chickens in Northern Vietnam.

Newcastle disease (ND) is a major viral disease of poultry worldwide. However, data on the molecular characterization of Newcastle disease virus (NDV) in Vietnam are limited. This study aimed to identify the molecular characteristics of NDV strains from the vaccinated chickens farmed in Northern Vietnam. We used reverse-transcription polymerase chain reaction (PCR), sequencing and phylogenetic analysis to characterize NDV strains from vaccinated chicken farms in Northern Vietnam. Seven out of 72 (9.7%) chicken tissue samples collected from seven chicken farms in the four cities/provinces in northern Vietnam were positive for the NDV genome by PCR method. The complete sequences of the fusion (F) and hemagglutinin-neuraminidase (HN) genes of NDVs isolated in the North of Vietnam from 2021 to 2022 were further evaluated. The results indicated that all seven Vietnamese isolates obtained were reported as virulent NDV strains with the amino acid (AA) sequence of the F0 protein proteolytic cleavage site motif (112RRRKRF117). Phylogenetic analysis revealed that they were grouped with other NDV class II from subgenotype VII.2, including the two previous Vietnamese NDV (2015), the Chinese (2017), and Southern African (2013) NDV strains. In addition, some AA substitutions were observed in the neutralizing epitopes of the F and HN proteins of the current Vietnamese NDV strains. The present findings provide useful information for future studies of the evolution of NDVs and improve strategies for ND-controlling programs in Vietnam.

Genome-wide Identification of HvPP2C Genes and Expression Profiling in Response to Cold and Heat Stresses in Barley (Hordeum vulgare L.)

Background: Protein phosphatases (PP2Cs) are the main classes of protein phosphatases in plants, having pivotal functions in different plant stages and abiotic stresses. The PP2C genes are suggested to have critical roles in barley by exposure to heat and cold treatments. Objectives: We surveyed HvPP2C genes from the barley genome in the present study. Phylogenetic analysis, functional annotation, synteny analysis, chromosomal distribution, prediction of cis-elements, and gene expression of HvPP2Cs under abiotic stresses were studied. Methods: In this study, HvPP2Cs of barley were surveyed using bioinformatics tools, and HvPP2C gene expression profiles under cold and heat stresses in 3 tissues (root, leaf, and stem) were analyzed. Results: In this study, 61 HvPP2C genes were detected from barley, and a phylogenetic tree was divided into 13 subfamilies. The results of chromosomal distribution showed that the HvPP2C genes were located on 7 chromosomes. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis of HvPP2Cs showed that they were largely expressed in different tissues (root, stem, and leaf) in the Azaran and Jolge barley cultivars. In Azaran, the HvPP2C gene expression increased in response to cold stress, whereas, in the Jolge cultivar, the HvPP2C gene expression increased in response to heat stress. The analysis of synteny revealed that HvPP2C24b with HvPP2C24a, HvPP2C24 with HvPP2C24a and HvPP2C24b; HvPP2C5 with HvPP2C5a, and HvPP2C41 with HvPP2C41a were paralogous. Conclusions: Results revealed a broad understanding of the HvPP2C gene family in barley, which can be valuable for the functional description of HvPP2Cs in plant response to abiotic stresses.

Insect cell‐derived human CD200‐Fc increases zonula occludens‐1 tight junction protein in urothelial carcinoma cells

AbstractCD200 is a ligand that interacts with the CD200 receptor 1, with an anti‐inflammatory effect. CD200 inhibits the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathway, attenuating the inflammatory response and maintaining barrier function. In this study, human CD200 was fused with the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) to generate the recombinant CD200‐Fc protein, produced in insect cells using the baculovirus expression vector system. The CD200‐Fc gene was cloned under the control of the polyhedrin promoter in the pFastBac‐1 vector of the baculovirus expression vector system. Immunoblot analysis confirmed the expression of CD200‐Fc in insect cells. The CD200‐Fc protein was successfully purified using protein A affinity chromatography. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay revealed that purified CD200‐Fc protein inhibited the proliferation of TCCSUP cells, a bladder epithelial cancer cell expressing CD200 receptor 1, at concentrations of 1, 10 and 100 ng/mL. Quantitative real‐time reverse transcription PCR (qRT‐PCR) and immunoblot analyses confirmed that the mRNA and protein levels of zonula occludens‐1, a tight junction protein for barrier protection in epithelial tissues, were increased in TCCSUP cells treated with insect cell‐derived CD200‐Fc. These results suggest that insect cell‐derived CD200‐Fc could alter zonula occludens‐1 expression in bladder epithelial cancer cells, enhancing the function of the cell barrier protein, which could inhibit cancer metastasis. Taken together, the baculovirus expression vector system can be applied to express antitumor therapeutic CD200‐Fc protein for the inhibition of bladder cancer.

A novel autosomal dominant ERLIN2 variant activates endoplasmic reticulum stress in a Chinese HSP family.

Hereditary spastic paraplegia (HSP) has been reported rarely because of a monoallelic variant in ERLIN2. The present study aimed at describing a novel autosomal dominant ERLIN2 pedigree in a Chinese family and exploring the possible mechanism of HSP caused by ERLIN2 variants. The proband and his family underwent a comprehensive medical history inquiry and neurological examinations. Whole-exome sequencing was performed on the proband, and Sanger sequencing was performed on some family members. HeLa cell lines and mouse primary cortical neurons were used for immunofluorescence (IF) and reverse transcription-PCR (RT-PCR). Seven patients were clinically diagnosed with pure spastic paraplegia in four consecutive generations with the autosomal dominant inheritance model. All patients presented juvenile-adolescent onset and gradually worsening pure HSP phenotype. Whole-exome sequencing of the proband and Sanger sequencing of all available family members identified a novel heterozygous c.212 T>C (p.V71A) variant in exon 8 of the ERLIN2 gene. The c.212 T>C demonstrated a high pathogenic effect score through functional prediction. RT-PCR and IF analysis of overexpressed V71A revealed an altered ER morphology and increased XBP-1S mRNA levels, suggesting the activation of ER stress. Overexpression of V71A in primary cultured cortical neurons promoted axon growth. The novel c.212 T>C heterozygous variant in human ERLIN2 caused pure HSP. Moreover, c.212 T>C heterozygous variant in ERLIN2 increased ER stress and affected axonal development.

Silencing of Glutamine: Fructose-6-Phosphate Aminotransferase Impairs Growth and Development in Sogatella furcifera (Hemiptera: Delphacidae).

Glutamine: fructose-6-phosphate aminotransferase (GFAT), the fourth enzyme in the chitin synthesis pathway, exerts wide-ranging effects on the growth and development of organisms. However, the role of GFAT in Sogatella furcifera remains unknown. In this study, the functional significance of the GFAT gene of S. furcifera was analyzed using a reverse transcription-polymerase chain reaction and RNA interference (RNAi) analyses. The complementary DNA sequence of SfGFAT was 3162 bp in length and contained a 2067 bp open reading frame encoding 688 amino acid residues. Structural domain analysis indicated that the SfGFAT protein consisted of one glutamine aminotransferase class 2 domain and two sugar isomerase domains. Expression profile analysis revealed that SfGFAT was expressed throughout the egg, nymph, and adult phases and was strongly expressed on the first day of each nymph stage and in the integuments of five tissues. RNAi results revealed that SfGFAT gene silencing significantly inhibited the mRNA expression of the target gene and resulted in severe mortality among S. furcifera. In summary, these findings demonstrate that SfGFAT plays a critical role in the development of S. furcifera. Moreover, these results may aid in the development of methods to control the spread of S. furcifera.

Transcriptome profiling reveals transcriptional regulation of Protegrin-1 on immune defense and development in porcine granulosa cells

Protegrin-1 (PG1) is an antimicrobial peptide (AMP) that has garnered increasing attention due to its potent immune defense activity. Our previous studies demonstrated the ability of PG1 to enhance proliferation and inhibit apoptosis of porcine granulosa cells (GCs) under oxidative stress. GCs play a crucial role in ovary follicular development. However, the specific function and underlying mechanisms of AMP in follicular development still need further elucidation. The present study aimed to comprehensively explore the biological effects of PG1 on porcine GCs using transcriptome profiling by RNA sequencing technology. Isolated GCs were incubated with or without PG1 for 24 h and transcriptome-wide analysis was exerted to identify differentially expressed genes (DEGs). The results of expression analysis revealed 1,235 DEGs, including 242 up-regulated genes and 993 down-regulated genes (|log2 (FoldChange)| > 1; adjusted P-value < 0.05). The expression levels of 7 selected DEGs were validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis, which was consistent with the RNA-sequencing data. Among the significant DEGs, several genes associated with GC function and ovarian follicle development were identified, such as estrogen receptor 2 (ESR2), growth and differentiation factor 6 (GDF6), cell division cycle 20 homolog (CDC20), Notch3, ephrin and Eph receptor system, Egl nine homolog 3 (EGLN3), and BCL2 like 14 (BCL2L14). Gene Ontology (GO) analysis revealed that the top three significant GO terms were inflammatory response, defense response, and granulocyte migration. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis presented that DEGs were mainly enriched in the immune system, infectious disease, signaling molecules and interaction, and immune disease. Furthermore, Ingenuity Pathway Analysis (IPA) predicted that the top activated pathway was Liver X Receptor (LXR)/ Retinoid X Receptor (RXR) Activation which is known to be associated with female reproduction. Predicted protein–protein interactions (PPIs) analysis identified complement C3 (C3) as the top node with the highest degree of network connection and revealed that DEGs in the sub-networks were involved in cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, chemokine signaling pathway, and metabolic process. In conclusion, this study expanded the understanding of the effects of PG1 on porcine GCs at the transcriptomic level and provided a theoretical basis for further investigation into the role of PG1 in immune defense and mammalian ovarian follicular development.

Exposure to sublethal concentrations of thiacloprid insecticide modulated the expression of microRNAs in honeybees (Apis mellifera L.)

This study aimed to investigate the sublethal effects of thiacloprid on microRNA (miRNA) expression in honeybees (Apis mellifera L.) and the role of a specific miRNA, ame-miR-283–5p, in thiacloprid resistance. The high-throughput small RNA-sequencing was used to analyze global miRNA expression profile changes in honeybees orally exposed to sublethal concentrations of thiacloprid (20 mg/L and 4 mg/L) for 72 h. Thiacloprid at 20 mg/L had no observed adverse effects. In addition, bees were fed with miRNA mimics or inhibitors to increase or decrease ame-miR-283–5p expression, and its effects on P450 gene expression (CYP9Q2 and CYP9Q3) were examined. Thiacloprid susceptibility was also detected. The results showed that treatment with thiacloprid at 20 mg/L and 4 mg/L induced 11 and five differentially expressed miRNAs (DEMs), respectively. Bioinformatic analysis suggested that the DEMs are mainly involved in the regulation of growth and development, metabolism, nerve conduction, and behavior. ame-miR-283–5p was downregulated by both concentrations, which was validated using quantitative real-time reverse transcription PCR analysis. Enhancing ame-miR-283–5p expression significantly inhibited CYP9Q2 mRNA and protein expression, and increased thiacloprid toxicity, while reducing ame-miR-283–5p expression significantly promoted CYP9Q2 expression, and decreased thiacloprid susceptibility. Our study revealed a novel role of miRNAs in insecticide resistance in honeybees.

The BioCascade-VIVAS system for collection and delivery of virus-laden size-fractionated airborne particles

The size of virus-laden particles determines whether aerosol or droplet transmission is dominant in the airborne transmission of pathogens. Determining dominant transmission pathways is critical to implementing effective exposure risk mitigation strategies. The aerobiology discipline greatly needs an air sampling system that can collect virus-laden airborne particles, separate them by particle diameter, and deliver them directly onto host cells without inactivating virus or killing cells. We report the use of a testing system that combines a BioAerosol Nebulizing Generator (BANG) to aerosolize Human coronavirus (HCoV)-OC43 (OC43) and an integrated air sampling system comprised of a BioCascade impactor (BC) and Viable Virus Aerosol Sampler (VIVAS), together referred to as BC-VIVAS, to deliver the aerosolized virus directly onto Vero E6 cells. Particles were collected into four stages according to their aerodynamic diameter (Stage 1: >9.43 μm, Stage 2: 3.81–9.43 μm, Stage 3: 1.41–3.81 μm and Stage 4: <1.41 μm). OC43 was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses of samples from all BC-VIVAS stages. The calculated OC43 genome equivalent counts per cm3 of air ranged from 0.34 ± 0.09 to 70.28 ± 12.56, with the highest concentrations in stage 3 (1.41–3.81 μm) and stage 4 (<1.41 μm). Virus-induced cytopathic effects appeared only in cells exposed to particles collected in stages 3 and 4, demonstrating the presence of viable OC43 in particles <3.81 μm. This study demonstrates the dual utility of the BC-VIVAS as particle size-fractionating air sampler and a direct exposure system for aerosolized viruses. Such utility may help minimize conventional post-collection sample processing time required to assess the viability of airborne viruses and increase the understanding about transmission pathways for airborne pathogens.

Heterogeneity characterization of hepatocellular carcinoma based on the sensitivity to 5-fluorouracil and development of a prognostic regression model.

Background: 5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in clinical cancer treatment, including hepatocellular carcinoma (HCC). A correct understanding of the mechanisms leading to a low or lack of sensitivity of HCC to 5-FU-based treatment is a key element in the current personalized medical treatment. Methods: Weighted gene co-expression network analysis (WGCNA) was used to analyze the expression profiles of the cancer cell line from GDSC2 to identify 5-FU-related modules and hub genes. According to hub genes, HCC was classified and the machine learning model was developed by ConsensusClusterPlus and five different machine learning algorithms. Furthermore, we performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis on the genes in our model. Results: A total of 19 modules of the cancer cell line were divided by WGCNA, and the most negative correlation with 5-FU was the midnight blue module, from which 45 hub genes were identified. HCC was divided into three subgroups (C1, C2, and C3) with significant overall survival (OS) differences. OS of C1 was the shortest, which was characterized by a high clinical grade and later T stage and stage. OS of C3 was the longest. OS of C2 was between the two subtypes, and its immune infiltration was the lowest. Five out of 45 hub genes, namely, TOMM40L, SNRPA, ILF3, CPSF6, and NUP205, were filtered to develop a risk regression model as an independent prognostic indicator for HCC. The qRT-PCR results showed that TOMM40L, SNRPA, ILF3, CPSF6, and NUP205 were remarkably highly expressed in hepatocellular carcinoma. Conclusion: The HCC classification based on the sensitivity to 5-FU was in line with the prognostic differences observed in HCC and most of the genomic variation, immune infiltration, and heterogeneity of pathological pathways. The regression model related to 5-FU sensitivity may be of significance in individualized prognostic monitoring of HCC.