qPCR Analysis Research Articles

Tyrosine Hydroxylase Is Required for the Larval-Pupal Transformation and Immunity of Plutella xylostella: Potential for Pest Management.

Plutella xylostella has developed high levels of resistance to many commonly used insecticides. Tyrosine hydroxylase (TH) is essential for insect survival; thus, we evaluated whether TH could be a potential target for controlling P. xylostella. In this study, PxTH was identified; further qPCR analysis showed that PxTH increased its expression during larval pupation and was highly expressed in the head and epidermis of prepupa in P. xylostella. Subsequently, we found a significant decrease in insect pupation and eclosion rates after injection of dsPxTH or a feeding diet supplemented with 3-iodo-tyrosine (3-IT) as a TH inhibitor in P. xylostella. Moreover, this study suggested that PxTH enzyme activity and dopamine concentrations were significantly decreased, agreeing with the blockage of larval-pupal cuticle tanning, with thinner puparium and less melanization after feeding 3-IT. In addition, expression levels of four antimicrobial peptide genes were significantly inhibited after P. xylostella feeding with 3-IT, and injection of Escherichia coli resulted in 73.3% mortality, indicating that PxTH was required for immune responses. In summary, these results confirmed that PxTH was involved in the development and immunity of P. xylostella, suggesting a critical potential novel insecticide target for RNAi-based pest control.

Establishment of Nile Tilapia Primary Cell Culture Methods and In Vitro Cell Knockdown Techniques.

As an important aquaculture species and research model, Nile tilapia (Oreochromis niloticus) has not yet been systematically studied for the isolation, culture, and in vitro gene manipulation techniques of primary cells from various tissues. This study aimed to explore methods for isolating primary cells from various tissues, as well as developing in vitro gene manipulation techniques in Nile tilapia. Four different Nile tilapia tissues were enzymatically digested and separated using trypsin or collagenase. Collagenase (0.1%) was used for the digestion of the gonads, liver, and heart, while trypsin (0.25%) showed better adhesion efficiency for spleen tissue. Moreover, we assessed EGFP fluorescence intensity and cell survival rates following transfection with empty siRNA (siRNA-NC), lentivirus (LV-NC), and six adeno-associated virus (AAV-NC) serotypes (AAV2-NC, AAV5-NC, AAV6-NC, AAV8-NC, AAV9-NC, AAV-DJ-NC) in gonadal cells. The results demonstrated that cells transfected with siRNA-NC and LV-NC showed the highest levels of green fluorescent protein expression and survival rates in primary gonadal cells, compared to AAC-NC. Subsequently, we knocked down the Kdm6bb gene in Nile tilapia primary gonadal cells by transfecting them with LV-Kdm6bb and siRNA-Kdm6bb. qPCR and immunofluorescence analyses demonstrated a significant reduction in Kdm6bb mRNA levels following transfection with siRNA-Kdm6bb compared to siRNA-NC, and with LV-Kdm6bb compared to LV-NC. This study offers valuable tools for the validation of primary cell isolation and in vitro molecular regulatory mechanisms and functions in Nile tilapia.

Potential radiosensitive germline biomarkers in the testes of wild mice after the Fukushima accident

We investigated potential germline‐specific radiosensitive biomarkers in the testes of large Japanese field mice (Apodemus speciosus) exposed to low‐dose‐rate (LDR) radiation after the Fukushima accident. Fukushima wild mice testes were analysed via RNA‐sequencing to identify genes differentially expressed in the breeding and non‐breeding seasons when compared to controls. Results revealed significant changes during the breeding season, with Lsp1 showing a considerable upregulation, while Ptprk and Tspear exhibited significant reductions. Conversely, in the non‐breeding season, Fmo2 and Fmo2 (highly similar) were significantly upregulated in radiation‐exposed Fukushima mice. qPCR analysis results were consistent with transcriptome sequencing, detecting Lsp1 and Ptprk regulation in the testes of Fukushima mice. While differences in gene expression were observed, these do not imply any causal association between the identified biomarkers and chronic LDR exposure, as other factors such as the environment and developmental age may contribute. This study provides valuable insights into the reproductive biology is affected by environmental radiation and highlights the value of assessing the effects of chronic LDR radiation exposure on testicular health in wild mice.

Effect of Perilla frutescens polyphenols against tight junction alterations and intestinal inflammation: a Caco-2/macrophage co-culture model-based study integrated with network pharmacology

The intestinal barrier plays important roles in the uptake of nutrients and prevention of harmful pathogens. However, intestinal inflammation causes barrier dysfunction, which may contribute to inflammatory bowel disease (IBD). Polyphenols from Perilla frutescens (L.) Britt. exhibit various bioactivities, though their protective effect on the intestinal barrier remains unclear. To address this, the study investigated the effect of Perilla frutescens (L.) Britt. polyphenols (PFP) against tight junction alterations and intestinal inflammation using an intestinal-like Caco-2/macrophage co-culture model. LC-MS/MS analysis identified 15 polyphenols in the PFP extract. Furthermore, PFP were transported across the apical Caco-2 monolayer, with apigenin, genistin, syringic acid and cinnamic acid showing relatively high transport rates (> 70%). The lipopolysaccharide (LPS)-induced tight junction alterations, evaluated by trans-epithelial electrical resistance (TEER) analysis, qPCR, and immunofluorescence assay, were significantly restored by medium and high doses of PFP (P < 0.05). In addition, network pharmacology analysis demonstrated that the core targets of PFP for intestinal inflammation were mostly located in the MAPK and NF-κB signaling pathways. The expression levels of the key proteins in these pathways were further validated by qPCR and western blot analyses. Overall, PFP exhibits great potential as a novel functional food component for treating intestinal inflammation.

The effect of antibacterial peptide ε-Polylysine against Pseudomonas aeruginosa biofilm in marine environment

Natural antibacterial agents with antimicrobial properties have a broad potential to prevent bacterial from forming biofilms adhesion in marine environments. ε-Polylysine (E-PL) consist of homomeric polymer with 25–30 lysine residues with stability, nontoxicity, and biodegradability. ε-Polylysine is a natural cationic antibacterial peptide that can resist microbial forming biofilm adhesion. The current study investigated the action of E-PL against Pseudomonas aeruginosa biofilm isolated from a marine environment. Crystal violet staining was used to examine the effects of E-PL on the formation and destruction of mature biofilms. Scanning Electron and fluorescence microscopy revealed that E-PL treatment damaged the biofilm structure and affected the secretion of extracellular polymers. The CCK8 colorimetric assay showed that E-PL also decreased the metabolic activity and motility of biofilm bacteria. QPCR and transcriptome analysis revealed that E-PL affected biofilm formation and transcriptional regulation by downregulating genes involved in flagellar synthesis (flgE, PA4651, pilW), chemotaxis transduction (PA1251, PA4951, PA4788), biofilm biosynthesis (pelC, pelD, pslK, plsM), transcriptional regulation (PA3973, PA3508, PA0268), phenazine biosynthesis (phzM, phzH, phzS), and electron transfer (PA5401, PA5400, PA3492). This study used multiple methods to identify the mechanism of E-PL action against biofilm, informing the design of novel biofilm treatment methods.

Comprehensive Analysis of circRNA and mRNA Revealing Potential Mechanism Underlying Neuroinflammation in BV2 Cells

Background: The significance of circular RNAs (circRNAs) in diabetic complications has been established. However, their role in basal and diabetic states, as well as cognitive dysfunction, requires further investigation. Methods: BV-2 microglial cells were exposed to high glucose (50mM) and insulin (2μM) for 48 hours. The levels of interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factoralpha (TNF-α) were assessed through quantitative polymerase chain reaction (qPCR), western blot, and ELISA. CircRNA and messenger RNA (mRNA) sequencing were performed, and the data were analyzed. Differentially expressed circRNAs and mRNAs were identified using qPCR. The circRNA-miRNA interaction was predicted using Miranda and TargetScan software, and their levels were quantified by qPCR. Results: The results demonstrated a significant increase in mRNA and protein levels of IL-1β, IL-6, and TNF-α in BV2 cells treated with glucose and insulin. Five circRNAs (four upregulated and one downregulated) were identified in both glucose and insulin groups compared to the control. Further qPCR analysis revealed marked increases in the levels of chr17:40159331- 40159711+ and chr2:72800499-72801858- (mmu_circ_0010164) in both treatment groups. Competitive endogenous RNA networks showed significant upregulation of mRNA levels of mitochondrial transcription termination factor 1b (Mterf1b) and G protein subunit gamma 4 (Gng4), accompanied by a decrease in mmu-miR-6918-3p and mmu-miR-7043-3p levels in the glucose and insulin groups compared to the control. Knockdown of mmu_circ_0010164 significantly inhibited the inflammatory response induced by glucose and insulin in BV-2 microglial cells. Conclusion: These findings indicate that both glucose and insulin can elicit inflammatory responses in BV2 cells through the modulation of mmu_circ_0010164 levels. The underlying mechanism may involve potential downstream targets of mmu_circ_0010164, specifically mmu-miR-7043-3p/Gng4 and mmu-miR-6918-3p/Mterf1b. This provides novel insights into the treatment of glucose-induced neuroinflammation.

Randomized Clinical Trial Evaluating a Novel Piperazine-Based Drug as a Treatment for Gastric Cryptosporidiosis in Eastern Indigo Snakes (Drymarchon couperi)

Abstract Gastric cryptosporidiosis caused by Cryptosporidium serpentis has impacted the conservation efforts for the imperiled eastern indigo snake (EIS, Drymarchon couperi). Multiple treatments, including paromomycin, nitazoxanide-azithromycin-rifabutin, clofazimine, curcumin, and hydrogen peroxide gavage, have been investigated and proved ineffective in eliminating the parasite, necessitating further investigation. A novel piperazine-based compound, MMV665917, has been shown to be more effective than paromomycin, nitazoxanide, and clofazimine in treating cryptosporidiosis in mammals. With these promising results, MMV665917 was selected to investigate as a treatment of gastric cryptosporidiosis in EIS. Twelve EIS naturally infected with C. serpentis were randomly divided into two groups of six: Group A and Group B. Group A received 22 mg/kg MMV665917 daily for 7d and Group B received a placebo daily for 7d. All EIS tolerated the treatment with no adverse effects a ppreciated. Success was evaluated by C. serpentis-specific probe hybridization qPCR analysis of gastric lavage performed one, two, and three months following the treatment. Infections persisted following treatment and there was no statistical difference in qPCR CT values between the groups or within the groups for the three months following treatment (F = 0.92, P = 0.525). These findings show that MMV665917 dosed at 22 mg/kg daily for 7d is ineffective in eliminating C. serpentis in EIS and alternative MMV665917 dosing or formulations should be investigated in reptiles.

H3K27 trimethylation-mediated downregulation of miR-216a-3p in sensory neurons regulates neuropathic pain behaviors via targeting STIM1.

Although the therapeutic potential of miRNA-mediated gene regulation has been investigated, its precise functional regulatory mechanism in neuropathic pain remains incompletely understood. In this study, we elucidate that miR-216a-3p serves as a critical non-coding RNA involved in the modulation of trigeminal-mediated neuropathic pain. By conducting RNA-seq and qPCR analysis, we observed a notable decrease of miR-216a-3p in the injured trigeminal ganglia (TG) of male rats. Intra-TG administration of miR-216a-3p agomir or lentiviral-mediated overexpression of miR-216a-3p specifically in sensory neurons of injured TGs alleviated established neuropathic pain behaviors, while downregulation of miR-216a-3p (pharmacologically or genetically) in naïve rats induced pain behaviors. Moreover, nerve injury significantly elevated the H3K27 trimethylation (H3K27me3) levels in the ipsilateral TG, thereby suppressing the SRY-box transcription factor 10 (SOX10) binding to the miR-216a-3p promoter and resulting in the reduction of miR-216a-3p. Inhibiting the enzymes that responsible for catalyzing H3K27me3 restored the nerve injury-induced reduction in miR-216a-3p expression and markedly ameliorated neuropathic pain behaviors. Furthermore, miR-216a-3p targeted stromal interaction molecule 1 (STIM1), and the decreased miR-216a-3p associated with neuropathic pain caused a significant upregulation in the protein abundance of STIM1. Conversely, overexpression of miR-216a-3p in the injured TG suppressed the upregulation of STIM1 expression and reversed the mechanical allodynia. Together, the mechanistic understanding of H3K27me3-dependent SOX10/miR-216a-3p/STIM1 signaling axial in sensory neurons may facilitate the discovery of innovative therapeutic strategies for neuropathic pain management.Significance Statement miRNAs are posttranscriptional regulators of gene expression that play critical roles in the pathogenesis of neuropathic pain. However, the detailed mechanisms by which most pain-associated miRNAs operate and their therapeutic potential are incompletely understood. Our present study revealed that nerve injury-induced trimethylation of lysine 27 on histone H3 (H3K27me3) reduces the binding of SOX10, a transcription factor, to the promoter region of the miR-216a-3p gene, leading to decreased expression of the microRNA, miR-216a-3p. This reduction subsequently promotes neuropathic pain by regulating STIM1. Given that miRNA-mediated gene regulation is a proposed therapeutic approach for treating neuropathic pain, our findings suggest that replenishing miR-216a-3p could serve as a novel strategy for treating chronic neuropathic pain.

Effect of Cow Bone Addition on the Humification, Heavy Metals Passivation and Fate of Resistance Genes During Swine Manure Composting

Bone meal has been used as economic and effective additive for heavy metals (HMs) pollution remediation due to the distinct components and structures that enable their favorable properties, such as its low cost, high adsorption capacity, acid-base adjustability, and ion-exchange capability. However, no attempt has been made to establish whether cow bone could promote the passivation of HMs and the removal of metal resistance genes (MRGs) and antibiotics resistance genes (ARGs) during the composting process. Two sizes of cow bone (meal (T2) and granule (T3)) were added to investigate their effects on humification, HMs passivation and the abundance of ARGs and MRGs during swine manure composting. Excitation-emission matrix (EEM)-parallel factor analysis showed that the percentage of maximum fluorescence intensity of humic-like substances were higher in T2 (91.82%) than in T3 (88.46%), implying that T2 could promote the humification process compared to T3. In comparison with control (T1), the addition of T2 and T3 could promote the change of exchangeable Cu and reducible Cu into oxidizable Cu, thus reducing the mobility factors (MF) of Cu in T2 and T3 treatments by 10.48% and 6.98%, respectively. In addition, T2 and T3 could increase exchangeable Zn into reducible Zn and oxidizable Zn, thereby reducing the MF of Zn in T2 and T3 treatments by 18.80% and 2.0%, respectively. Quantitative Real-time PCR (qPCR) analysis revealed that the total abundances of MRGs were decreased by 100% in T2 and T3 treatments, and T2 decreased the total relative abundance of ARGs. Furthermore, the relative abundance of ARGs and MRGs had significantly correlated with intI1 and bio-available of Cu and Zn, which was triggered by selective pressure of HMs and horizontal gene transfer. The present study suggested that cow bone meal as additives can be a feasible approach to promote the passivation of HMs and enhance the removal of MGRs and ARGs by decreasing horizontal gene transfer and selective pressure by bioavailable HMs.

Anticariogenic activity of marine brown algae Padina boergesenii and its active components towards Streptococcus mutans

Streptococcus mutans is a well-recognized bacterium that plays a predominant role in the progression of dental caries. Its pathogenicity is linked to several key characteristics, including the ability to produce organic acids (acidogenicity), thrive in low pH environments (aciduricity), synthesize exopolysaccharides (EPS) via glucosyltransferases, and form retentive biofilms. The treatment of dental caries with conventional antibiotics is often ineffective due to the bacterium’s capacity to form recalcitrant biofilms. To address these challenges, strategies that specifically target the pathogen’s virulence without affecting its viability have emerged as promising alternatives. In this context, we investigated the anticariogenic properties of the methanolic extract of Padina boergesenii (MEPB). MEPB demonstrated substantial, dose-dependent antibiofilm activity, with a maximum inhibition of 93% at 128 μg/mL, without compromising the viability of S. mutans. Anti-virulence assays using sub-MIC (minimum inhibitory concentration) levels of MEPB showed significant reductions in key virulence factors: 75% reduction in sucrose-dependent adherence, 65% reduction in sucrose-independent adherence, along with notable decreases in acid production, acid tolerance, and water-insoluble (85%) and water-soluble (52%) glucan synthesis. Additionally, MEPB significantly reduced cell surface hydrophobicity (55%) and extracellular DNA (eDNA) production (64%). qPCR analysis corroborated these in vitro findings, revealing that MEPB suppresses the expression of genes involved in S. mutans virulence, particularly genes related to EPS synthesis (gtfB, gtfC &amp;amp; gtfD) biofilm formation(gbpB &amp;amp; gbpC) and two-component regulatory system (vicR) were downregulated. Toxicity testing on human buccal epithelial cells confirmed the non-toxic nature of MEPB, suggesting its safety for potential therapeutic use. Furthermore, GC-MS/MS analysis identified palmitic acid, myristic acid, and stearic acid as the major active constituents of the MEPB extract. Subsequent biofilm inhibitory assays confirmed the potent antibiofilm efficacy of these compounds: palmitic acid (85%), myristic acid (72%) and stearic acid (83%). In conclusion, this study identifies P. boergesenii and its active biomolecules as potential anticariogenic agents, offering an alternative approach to combat dental caries by targeting bacterial virulence mechanisms rather than viability.

Molecular detection of haemobacteria in Colombian wild birds

Colombia shows a high density and variety of bird species, making it one of the most diverse avian territories globally. Antioquia ranks among the top four provinces with the greatest variety of bird species, underscoring the importance of research efforts on the local bird fauna. Therefore, this study aimed to identify bacterial agents in the blood of wild birds from the municipality of Jardín (Antioquia, Colombia) by 16S qPCR sequencing. A descriptive cross-sectional study was conducted using non-probabilistic convenience sampling. Wild birds were captured using mist nets and blood samples were collected from each animal via puncture using sterile lancets in the brachial vein, and a drop of blood was collected on filter paper for qPCR analysis. The 16S gene in bacterial genomes was found in 13 out of 46 wild birds of the Passeriform and quasi-Passeriform orders, captured at three different locations within the study municipality at altitudes ranging from 1,665 to 2,034 m.a.s.l. Seven different bird species were recorded and four different haemobacteria were identified (i.e. Exiguobacterium spp., Escherichia coli, Stenotrophomonas spp., and Stenotrophomonas maltophilia). This study contributes to the knowledge in Colombia by identifying four different hemobacteria in wild birds. Further research is required on the health status of these birds and the attributable impacts on their populations and other related factors, including humans.

Fasting regulates expression of voltage-gated Na+ channel Nav1.3 in subfornical organ

The subfornical organ (SFO) is a sensory circumventricular organ of the central nervous system and plays a key role in regulation of a number of homeostatic processes because of its ability to detect and respond to circulating signals and communication to homeostatic control centres. A previous study reported a change in expression of 687 transcripts in rat SFO following a 48h fast; of particular interest was the observed downregulation of the transcript encoding the Nav1.3 voltage-gated Na+ channel. Therefore, we carried out a study to examine the effects of a 48h fast on electrical properties of SFO neurons. First, we carried out an immunohistochemical analysis of rat SFO to confirm expression of Nav1.3 protein. Next, we carried out qPCR analysis of mRNA from SFO of sated rats and 48h fasted rats and confirm that a 48hr fast caused a downregulation of Nav1.3. Using patch clamp analysis of SFO neurons acutely isolated from rats following a 48h fast, a statistically significant decrease in peak Na+ current density, as well as shifts in voltage dependence of activation and inactivation, and a slowing to time dependent recovery from inactivation were observed. These changes were accompanied by a depolarization of the threshold to fire action potentials and a decrease in frequency of spontaneous action potentials. Together, these data show that the electrical properties of SFO neurons are altered by a 48hr fast, indicating SFO is a dynamic sensor of circulating signals.

Reference gene selection for real-time qPCR in European flounder (Platichthys flesus) using organ-specific RNA-seq data

BackgroundThe European flounder is readily chosen as an experimental subject and model in physiological and ecotoxicological studies mostly because of its adaptability to laboratory conditions. Many studies utilise a quantitative PCR (qPCR) approach to ascertain the expression of target genes under experimental conditions. Such an approach relies heavily on the selection of reference genes with stable expression. Yet certain housekeeping genes are commonly used in this role, often without due consideration of their overall expression patterns. Therefore, new approaches should be developed to identify stable reference genes for a given species and to expand the general pool of genes suitable for the reference in qPCR analysis.Methods and resultsHere RNA-seq data of nine flounder organs led to identify four candidate genes of the most stable expression. It was achieved by differential expression analysis and tritoconstrictor script. Specific primers were designed for the complete ORF as well as for qPCR analysis. RT-qPCR efficiencies were tested on ORF amplicon templates. Most of the genes tested showed good amplification in a wide range of template dilutions (107-101), with a correlation coefficient (R2) ranging from 0.991 to 0.998 and a consistent efficiency (E) (Sybr Green I staining and TaqMan molecular probe).ConclusionsThe proposed approach based on differential expression analysis and a new bioinformatic tool is an appropriate selection method of candidates for reference genes in qPCR. The proposed approach, combining differential expression analysis with a new bioinformatics tool, provides an effective method for selecting reference gene candidates for qPCR. As a result, we can propose four genes (polr2f, yif1a, sf3b6, uba52), each with a set of validated primers, as suitable for consideration as reference genes in qPCR analysis in European flounder, an emerging model species.

Different infant formulas can activate toll-like receptor 9 in vitro and inhibit interleukin 6 in human primary intestinal epithelial cells

PurposeNecrotizing enterocolitis (NEC) is the most severe gastrointestinal disease in preterm infants caused by an exaggerated intestinal epithelial immune activation. Several studies show that modulation of toll-like receptor 9 (TLR9) activity may have positive effects on preventing intestinal inflammatory mechanisms ultimately resulting in NEC development. In this study, the effect of various infant formulas (IF) and the probiotic strain Limosilactobacillus fermentum CECT5716 on TLR9 activation were analyzed in vitro.MethodsFirst, TLR4 and TLR9 expression was analyzed on human primary intestinal epithelial cells (P-IECs) by qPCR and Western blot analysis. Then genetically designed HEK-Dual™ hTLR9 (NF/IL8) reporter cells (HEK-Dual) were treated with different IFs, L. fermentum CECT5716, and different functional components to measure TLR9 activation via luminescence. Finally, the IFs were investigated in P-IECs to analyze TLR downstream signaling by Western blot analysis.ResultsIFs containing intact protein and L. fermentum CECT5716 activated TLR9 in HEK-Dual cells, but the functional components lactoferrin, L-5-methyltetrahydrofolate, and hydrolyzed whey proteins failed to activate TLR9. In P-IECs, the IFs induced increased phosphorylation of MAPK8/9 of the TLR signaling pathway and significantly reduced IL6 levels. Consistently, IL6 levels were increased in P-IECs when TLR9-signaling was inhibited. Interestingly, L. fermentum CECT5716 enhanced TLR9-signaling and increased NF-kappa-B inhibitor alpha-phosphorylation.ConclusionWe found out that the used control formula, prebiotic formula, prebiotic formula with hydrolyzed-protein, and L. fermentum CECT5716 reduce IL6 levels in human P-IECs through TLR9 activation. L. fermentum CECT5716 and the here tested IFs could be a promising approach for modulation of gut health in infants.

Green hydrothermal synthesis of gallic acid carbon dots: characterization and cytotoxic effects on colorectal cancer cell line

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide, necessitating the development of novel therapeutic approaches. Carbon dots (CDs) have emerged as promising nanoparticles for biomedical applications due to their unique properties. Gallic acid (GA), an anticancer agent, is effective against various tumor types. This study explores the potential of gallic acid-derived carbon dots (GA-CDs) as an innovative anticancer agent against HCT-116 CRC cells, focusing on apoptosis signaling pathways. GA-CDs were synthesized using a one-pot hydrothermal method. Characterization was conducted using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and ultraviolet-visible (UV-vis) absorption spectroscopy. The cytotoxicity of GA and GA-CDs on HCT-116 cells was evaluated using the MTT assay at various concentrations over 24 and 48 h. Cellular uptake was assessed via fluorescence microscopy, and apoptosis was analyzed using acridine orange/propidium iodide (AO/PI) staining. Total RNA extraction followed by complementary DNA (cDNA) synthesis via reverse transcription-PCR was performed, and real time-PCR (Q-PCR) was conducted to examine the expression of apoptosis-related genes includingCaspase-3,Bax, andBcl-2. Characterization confirmed the successful synthesis of spherical GA-CDs. GA-CDs exhibited dose- and time-dependent cytotoxicity, with IC50 values of 88.55 μg ml-1for GA-CDs and 192.2 μg ml-1for GA after 24 h. Fluorescence microscopy confirmed the efficient uptake of GA-CDs by HCT-116 cells. AO/PI staining showed a significant increase in apoptotic cell numbers after treatment with GA-CDs. Q-PCR analysis revealed overexpression ofCaspase-3 andBaxgenes in GA-CD-treated cells, though no significant changes were observed in the expression ofBcl-2 or theBax/Bcl-2 ratio. GA-CDs demonstrated potent anticancer properties by inducing apoptosis and reducing cell viability in HCT-116 cells. These findings suggest the potential of GA-CDs as a novel therapeutic agent for CRC treatment, warranting further investigation into their mechanism of action andin vivoefficacy.

Genomic survey and evolution analysis of calcium-dependent protein kinases in plants and their stress-responsive patterns in populus

BackgroundCalcium-dependent protein kinases (CDPKs) phosphorylate downstream target proteins in response to signals transmitted by free calcium ions (Ca2+, one of the second messengers) and thus play important regulatory roles in many biological processes, such as plant growth, development, and stress response.ResultsA bioinformatic analysis, as well as thorough evolutionary and expression investigations, were conducted to confirm previous reports of functional evidence for plant CDPKs. Using the Phytozome database’s BLAST search engine and the HMM search tool in TBtools software, we discovered that CDPKs are well conserved from green algae to flowering angiosperms in various gene family sizes. Additional investigations of the obtained CDPKs revealed high conservation of domain and motif numbers, gene architectures, and patterns. However, this conservation differed among plant species. Phylogenetic analysis demonstrated that the CDPK gene family diverged from a common ancient gene. Similarly, investigations into plant interspecies evolutionary relationships revealed common ancestral plant species, suggesting speciation of plants and evolution based on plant adaptation and diversification. A search for the driving force of CDPK gene family expansion revealed that dispersed duplication events, among other duplication events, contributed largely to CDPK gene family expansion. Gene localization analysis in P. trichocarpa demonstrated that most CDPK genes are localized within several cell organelles and bind other kinases and proteins to perform their biological functions efficiently. Using RNA-seq data and qPCR analyses, we postulated that PtCDPKs play functional roles in abiotic stress responses by regulating cold, heat, drought and salt stress to varying extents.ConclusionThe CDPK genes are well conserved in plants and are critical entities in abiotic stress regulation, and further exploration and manipulation of these genes in the future may provide solutions to some of the challenges in agriculture, forestry and food security.

Unveiling genetic defense mechanisms: expression analysis of hym, AmNrx1, and CYP9Q3 genes in Varroa-resistant anatolian honey bees.

The Varroa destructor represents a significant threat to honey bees, leading to substantial yield losses and declines in colony health. Defense behaviors, including grooming (auto and allogrooming), serve as crucial mechanisms against Varroa infestations, yet the genetic basis of these behaviors remains elusive. This study examines the expression levels of hymenoptaecin (hym), neurexin-1 (AmNrx1), and CYP9Q3, potentially associated with defense behavior, in colonies of the Muğla honey bee ecotype (Apis mellifera anatoliaca) subjected to a Varroa selection program. Worker bees from 23 control groups and 23 colonies under selection were evaluated by using qPCR analysis. Results reveal a significant upregulation of hym, AmNrx1, and CYP9Q3 genes in the selected group, with fold changes of 2.9, 2.95, and 3.26 respectively compared to controls (p < 0.01). This suggests that selection against Varroa induces alterations in gene expression linked to Varroa exposure behaviors. These findings advocate for the potential use of hym, AmNrx1, and CYP9Q3 genes in preselection for future Varroa-resistant programs in honey bees. Supported by previous studies, these genes may facilitate the establishment of populations with enhanced defense behaviors, such as autogrooming and allogrooming.

Chamomile Tincture and Lidocaine Hydrochloride Gel Ameliorates Periodontitis: A Preclinical Study.

Background/Objectives: Periodontitis is a common oral disease marked by gingival inflammation and alveolar bone loss. This study evaluated the efficacy of chamomile tincture and lidocaine hydrochloride (CLH) gel in mitigating periodontal inflammation and bone loss and uncovered the molecular mechanisms involved, both in vitro and in vivo. Methods: A periodontitis model was induced in Sprague Dawley rats by ligating the mandibular first molars. Sixty rats were divided into four groups: control (C), periodontitis (PD), periodontitis + CLH gel once daily (G1), and periodontitis + CLH gel thrice daily (G3). Clinical, micro-computed tomography (micro-CT), biological, and histological evaluations were performed, focusing on osteoclastogenesis, osteogenesis, and inflammatory cytokine production. The effect of CLH gel on inflammatory responses in RAW264.7 cells was also assessed through co-culture assays under Porphyromonas gingivalis (P. gingivalis) infection, with RNA-sequencing, qPCR, and Western blot analyses to explore underlying mechanisms. Results: CLH gel significantly reduced gingival and systemic inflammation and mitigated bone loss by enhancing the bone volume to tissue volume ratio and trabecular thickness via the RANKL/OPG axis in rats. The G3 group showed marked reductions in osteoclasts and increases in osterix-positive cells compared to other groups. In vitro, CLH gel reduced the inflammatory phenotype of macrophages in the periodontitis microenvironment by modulating Type II interferon (IFN-γ) networks. Conclusions: CLH gel reduced inflammation and bone loss in rat periodontitis, promoting osteogenesis and inhibiting osteoclastogenesis. It also suppressed macrophage inflammation via Type II interferon networks under P. gingivalis stimulation. These findings suggest that CLH gel has potential as an adjunctive therapy for periodontitis.

Construction of a cell cycle-specific lncRNA-miRNA-mRNA network reveals novel key lncRNAs in colorectal cancer

Objective The current study aimed to determine the roles of pivotal and novel lncRNAs associated with the cell cycle in the occurrence and development of Colorectal cancer (CRC). Methods The TCGA-COAD project related to CRC was downloaded, and differential expression analysis was performed to identify differentially expressed lncRNAs, miRNAs, and mRNAs. A cell cycle-associated lncRNA-miRNA-mRNA regulatory network was constructed, and two novel lncRNAs were selected. Two subnetworks were constructed for selected lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were illustrated for the genes in each sub-network. qPCR analysis was used to validate the expression levels of the selected lncRNAs in CRC tissues compared to those adjacent normal tissues. Results The differential expression analysis identified 416 lncRNAs, 317 miRNAs, and 117 mRNAs. The ceRNA subnetwork genes were associated with different pathways, including cellular senescence, DNA replication, human T-cell leukemia virus 1 infection, and oocyte meiosis. The bioinformatic results based on the TCGA project indicated the dysregulation of two novel lncRNAs, MIR29B2CHG and HELLPAR, in CRC tissues compared to adjacent normal tissues. Moreover, qPCR confirmed the dysregulation of lncRNAs in the CRC tissues. ROC curves revealed that both selected lncRNAs had acceptable specificity and sensitivity as biomarkers. Conclusion In conclusion, novel cell cycle-associated lncRNAs have the potential to be understood as the underlying molecular mechanisms that influence CRC. Therefore, these lncRNAs can be considered as promising biomarkers for the diagnosis and treatment of CRC.

Modulation of metabolic pathways and EndMT inhibition by a traditional Chinese herbal formula in the treatment of high-risk infections.

Herbal products have long been utilized as remedies for various disease conditions, including infections. This study investigates the therapeutic mechanism of a traditional Chinese herbal combination in treating high-risk HPV infections. The herbal formula was prepared using common herbs: dry Millettia speciosa, Guanzhong (a spermatophyte), Sarsaparilla, White Fruit, and Cockscomb Flower. Eight female patients diagnosed with high-risk HPV were enrolled from January to September 2023 at Shenzhen Hospital of Beijing University of Traditional Chinese Medicine. Cervicovaginal secretions were collected before and after treatment with the herbal remedy and analyzed using non-targeted metabolomics techniques. In vitro studies were conducted using HeLa cells to determine the optimal effective concentration of the remedy, assessed via the CCK8 method. The proliferation and migration of HeLa cells were evaluated using Transwell assays. Quantitative PCR was employed to measure mRNA levels of endothelial-to-mesenchymal transition (EndMT) markers, including VE-Cadherin, eNOS, α-SMA, and Snail. In vivo, significant alterations in cervicovaginal secretion metabolites post-treatment were observed through PCA, OPLS-DA, and volcano plot analyses. KEGG enrichment analysis highlighted crucial signaling pathways such as arginine and proline metabolism, purine metabolism, glycerophospholipid metabolism, and phenylalanine metabolism, indicating the herbal combination's systemic effects on patients. In vitro experiments demonstrated a dose-dependent reduction in HeLa cell proliferation and migration, confirmed by scratch and Transwell assays. Additionally, qPCR analysis revealed down-regulation of α-SMA and Snail, and up-regulation of VE-Cadherin and eNOS, suggesting inhibition of EndMT in HeLa cells. The traditional Chinese herbal combination modulates key metabolic pathways in vivo and inhibits EndMT in vitro, while reducing HeLa cell proliferation and migration. These findings highlight its potential as a therapeutic approach for managing HPV infections, bridging traditional practices with scientific research.