qPCR Results Research Articles

Enhanced Synthesis of Volatile Compounds by UV-B Irradiation in Artemisia argyi Leaves

Background: Volatile compounds have a deep influence on the quality and application of the medicinal herb Artemisia argyi; however, little is known about the effect of UV-B radiation on volatile metabolites. Methods: We herein investigated the effects of UV-B exposure on the volatile compounds and transcriptome of A. argyi to assess the potential for improving its quality and medicinal characteristics. Results: Out of 733 volatiles obtained, a total of 133 differentially expressed metabolites (DEMs) were identified by metabolome analysis. These were classified into 16 categories, primarily consisting of terpenoids, esters, heterocyclic compounds, alcohols, and ketones. Sensory odor analysis indicated that green was the odor with the highest number of annotations. Among the 544 differentially expressed genes (DEGs) identified by transcriptome analysis, most DEGs were linked to “metabolic pathways” and “biosynthesis of secondary metabolites”. Integrated analysis revealed that volatiles were mainly synthesized through the shikimate pathway and the MEP pathway. RNA-seq and qPCR results indicated that transcription factors HY5, bHLH25, bHLH18, bHLH148, MYB114, MYB12, and MYB111 were upregulated significantly after UV-B radiation, and were therefore considered key regulatory factors for volatiles synthesis under UV-B radiation. Conclusions: These findings not only provide new insights into UV-induced changes in volatile compounds, but also provide an exciting opportunity to enhance medicinal herbs’ value, facilitating the development of products with higher levels of essential oils, flavor, and bioactivity.

Prevalence of Taylorella equigenitalis in Icelandic mares and geldings in Southern Germany and Austria

Contagious Equine Metritis (CEM) caused by the bacterium Taylorella equigenitalis (T. equigenitalis), is a venereal infection of equids which is of international concern to the equine breeding industry. A recent study showed a high prevalence of T. equigenitalis in Icelandic stallions when compared to stallions of other breeds also using for natural breeding. Consequently, the objectives of the present study were to investigate the prevalence of T. equigenitalis in Icelandic mares and geldings and to determine factors associated with a T. equigenitalis-positive qPCR result. In total, 361 Icelandic horses located in Southern Germany and Austria were tested for T. equigenitalis using a qPCR assay. An overall prevalence of 14.4% was detected. Positive qPCR results were found in 2.2% (3/134) of brood mares, 9.0% (11/122) of maiden mares and in 36.2% (38/105) of geldings. The odds for a T. equigenitalis-positive qPCR result were significantly lower in both brood (OR=40.1, 95% CI: 8.38-192, P<0.001) and maiden mares (OR=9.51, 95% CI: 3.26-25.7, P<0.001) when compared to geldings. Advancing age was not associated with higher odds for a T. equigenitalis-positive qPCR result (OR=0.98, 95% CI: 0.94-1.03, P=0.51). However, horses of the younger age group showed significantly lower Ct values compared to horses of the older age group (P=0.04). Furthermore, geldings showed significantly lower Ct values than brood (P<0.03) and maiden mares (P<0.001). This study showed a significantly higher prevalence of T. equigenitalis in Icelandic geldings compared to Icelandic mares. Icelandic geldings might therefore represent a reservoir for T. equigenitalis.

A Sensitive, Specific and Fast Electrochemical‐based Nanobiosensor Diagnostic for Xanthomonas albilineans, the Cause of Sugarcane Leaf Scald Disease

AbstractLeaf scald (LS) caused by Xanthomonas albilineans (Xalb), is a major bacterial disease of sugarcane. The unreliable symptom expressions make traditional visual detection challenging. The molecular methods of detection require expensive equipment, labor‐intensive, and time‐consuming. This study proposes a novel electrochemical (EC)‐approach, that is relatively easy to use and less expensive to detect Xalb DNA in LS‐infected sugarcane leaves, meristematic tissue, and xylem sap samples. This method involves three key steps: i) DNA isolation from sugarcane samples via boiling lysis; ii) magnetic purification of target sequences from the lysate using magnetic bead‐bound capture probes; and iii) EC detection of the target DNA. The method shows excellent detection sensitivity (10 cells µL−1), reproducibility (Standard deviation, SD &lt;5%, for n = 3), and a wide linear dynamic range (1 nM–1 fM or 106–10° copies µL−1, r = 0.99). The EC assay has a strong negative correlation with quantitative polymerase chain reaction (qPCR) results (r = −0.95–0.97, n = 24, p &lt; 0.001), and weak or no correlation with the varietal resistance ratings. This EC‐based assay can be a commercially viable alternative, providing a DNA isolation/purification‐free solution, and can potentially be adapted into a handheld device for on‐farm detection and quantification of the LS‐causing pathogen.

Enhanced anti-inflammatory effects and metabolic profiles of cyanidin-3-glucoside via nanoliposomal encapsulation in Caco-2 and RAW 264.7 co-culture system

Cyanidin-3-glucoside (C3G) has been found to prevent/treat intestinal disease and maintain gut homeostasis. Numerous studies have proved that nanoliposomes can improve the absorption and utilization of food nutrients. However, the impacts of C3G nanoliposomes (C3G-NL) are still unkown. This study investigated the effects of cyanidin-3-glucoside nanoliposomes (C3G-NL) on intestinal inflammation and permeability using a Caco-2/RAW264.7 co-culture model. ELISA and qPCR results showed that C3G-NL reduced pro-inflammatory markers, including TNF-α, IL-6, IL-8, cyclooxygenase-2, and inducible nitric oxide synthase, while inhibiting the NF-κB/MAPK pathway. C3G-NL also improved intestinal barrier integrity by increasing transepithelial electrical resistance and upregulating tight junction proteins (zonula occludens-1, occludin, claudin-1). Additionally, C3G-NL enhanced C3G metabolism, leading to the formation of intermediary metabolites like chalcone and dihydroflavonols, which contributed to the synthesis of other anthocyanins (delphinidin, peonidin, petunidin, malvidin, and pelargonidin). Overall, C3G-NL helps mitigate inflammation and restore tight junction integrity via metabolic regulation.

Transcriptional and post-transcriptional regulation of CARMN and its anti-tumor function in cervical cancer through autophagic flux blockade and MAPK cascade inhibition

BackgroundLncRNAs play essential roles in multiple tumors. However, research on genome-wide lncRNA alterations and their functions in cervical cancer (CC) is limited. This study aims to explore key lncRNAs in CC progression and uncover the molecular mechanisms involved in the development of CC.MethodsIn this study, we analyzed 30 tissues from CC, cervical intraepithelial neoplasia (CIN), and normal (NOR) using transcriptome sequencing and weighted gene co-expression network analysis to establish gene modules related to the NOR-CIN-CC transition. Machine learning diagnostic models were employed to investigate the role of lncRNAs in this transition. Molecular biological experiments were conducted to elucidate the potential mechanisms of CARMN in CC, with a particular focus on its transcriptional and post-transcriptional regulation of abnormal expression in CC.ResultsCARMN was identified as a hub gene in two modules significantly associated with the NOR-CIN-CC transition. Analysis using ten machine learning models confirmed its critical role in this progression. The results of RNA-seq, qPCR and RNAScope performed in another cohort of 83 cervical tissues all showed that CARMN was significantly downregulated in CC. CARMN significantly enhanced the interaction between Keap1 and Nrf2, leading to increased ROS levels. The elevated ROS levels suppressed the Akt/mTOR signaling pathway, leading to autophagy arrest via autophagic flux blockade. Additionally, CARMN interacted with TFAP2α to repress MAPK13 transcription, further inhibiting the MAPK cascade. A promoter SNP (rs12517403) was found to increase CC risk (OR = 1.34, 95% CI = 1.11–1.61) and reduce CARMN expression by decreasing SP1 binding. Furthermore, the RNA binding proteins that could modulate CARMN RNA stability were also determined using RNA-pulldown assay. The results demonstrated that YBX1, a component of the coding region instability determinant (CRD)-mediated mRNA stabilization complex, promoted CARMN RNA stability. DHX9, another component of complex, acted as a scaffold to bridge YBX1 and CARMN.ConclusionsCARMN exerts an anti-cancer effect in CC progression by inhibiting the Akt-mTOR and MAPK signaling pathways. rs12517403 and the YBX1/DHX9 complex are key mechanisms influencing its transcription and stability in CC cells. CARMN represents a promising biomarker for CC diagnosis and therapeutic target.

The Effect of Xanthohumol and Thymol on Candida albicans Filamentation and Its Impact on the Structure, Size, and Cell Viability of Biofilms Developed over Implant Surfaces.

The aim of this in vitro study was to evaluate the effect of xanthohumol and thymol on the impact of Candida albicans on the structure, size and cell viability of subgingival biofilms formed on dental implant surfaces. The structure and microbial biomass of biofilms developed after 72 h, treated and untreated with both extracts, were compared by scanning electron microscopy (SEM) and confocal laser microscopy (CLSM). Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total microorganisms of each of the biofilm-forming strains in each condition. A general linear model was used to compare and validate the CLSM and qPCR results. The presence of xanthohumol and thymol during biofilm development inhibited the filamentous growth of C. albicans. The biofilm incubated with xanthohumol had significantly lower bacterial biomass and cell viability than the biofilm not exposed to the extract (p < 0.05). In contrast, these global parameters showed no differences when the biofilm was incubated with thymol. In the presence of xanthohumol, there was a decrease in counts and cell viability of Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Thymol treatment reduced the viability of F. nucleatum and P. gingivalis. The presence of these vegetable extracts during the development of a dynamic in vitro multispecies biofilm model inhibited the filamentous growth of C. albicans, partially reversing the effect that the fungus exerted on the structure, size and vitality of periodontopathogenic bacteria.

Inter-Laboratory Comparison of qPCR Assays for Piscirickettsia salmonis in Atlantic Salmon (Salmo salar L.) in 11 Chilean Laboratories.

Real-time PCR (qPCR) testing is an essential component of early detection surveillance systems for Piscirickettsia salmonis infection in Atlantic salmon farms in Chile. Currently, all 11 laboratories in the authorised diagnostic laboratory network use assays based on published protocols. Compared with other P. salmonis qPCR assays, these assays have the advantage of targeting two different genes, that is, the 16S ribosomal gene and the internal transcribed spacer (ITS), potentially allowing for higher diagnostic accuracy. However, variation and lack of harmonisation of qPCR testing systems (e.g., primers/probe, RNA/DNA as target, extraction methods, etc.) may contribute to among-laboratory variation in qPCR results and an increased frequency of false-negative and false-positive results. The purpose of the ring trial reported herein was to compare qPCR results from 11 laboratories in Chile routinely testing Atlantic salmon for P. salmonis as part of a national control program. The panel of 14 samples included duplicates of three concentrations of P. salmonis in a homogenised head kidney, LF89 and EM90 bacteria and two negative controls (blank and a suspension of Flavobacterium psychrophilum). The sample order was randomised across labs, samples were tested blinded and analysed without knowledge of the source lab. Of the laboratories, 8 (72.7%) had at least one incorrect result out of 14 tested samples. Low-concentration samples (Ct of about 30) were more often incorrectly classified by reverse transcription-qPCR (RT-qPCR) (3/6 labs) than by qPCR (0/5). Six (54.5%) labs had at least one false-positive result indicating that cross-contamination was likely during sample processing. Affected laboratories are advised to conduct internal investigations to confirm the causes of false-positive results and recommendations for design and implementation of future ring trials are discussed.

D-mannose alleviates chronic periodontitis in rats by regulating the functions of neutrophils

BackgroundPeriodontitis is a chronic inflammatory disease characterized by the destruction of the components of the periodontium. It significantly impacts oral health and has been linked to systemic conditions like cardiovascular disease and diabetes. The critical role of neutrophils in the occurrence and development of chronic periodontitis has been paid increasing attention. The study aimed to explore the protective effects of D-mannose on chronic periodontitis and determine whether its underlying mechanisms is related to neutrophils.MethodsTo explore the protective effects of D-mannose on chronic periodontitis, the eight-week-old Sprague Dawley rat model of lipopolysaccharide (LPS)-induced periodontitis was established, followed by D-mannose treatment by oral gavage. To evaluate the protective effects of D-mannose against periodontal bone loss, methylene blue staining, hematoxylin and eosin (H&E) staining, and micro-CT scanning were utilized. Then, immunofluorescence (IF), Western Blot, and RT-PCR were applied to assess the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, and IL-17), anti-inflammatory cytokine (IL-10), tumor necrosis factor-alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), ten-eleven translocation 2 (TET2), and key glycolytic enzymes (HK1, HK2, PFKFB3), and to examine D-mannose’s impact on the recruitment and activation of neutrophils in the gingiva. Additionally, neutrophils isolated from the peripheral blood of healthy rats were treated with LPS and D-mannose, and changes in the expression levels of myeloperoxidase (MPO), IL-1β, IL-6, IL-17, IL-10, and TET2 were observed via IF.ResultsIn vivo, D-mannose inhibited LPS-induced alveolar bone resorption in rats. After D-mannose treatment, the expression levels of IL-17 (p<0.01) and TET2 (p<0.01) were suppressed by IF, and the expression levels of IL-1β (p<0.05), IL-17 (p<0.05) and TET2 (p<0.01) were downregulated by WB. The results of qPCR showed that D-mannose reduced the expression levels of IL-1β (p<0.05), IL-6 (p<0.01), IL-17 (p<0.01), TNF-α (p<0.01), G-CSF (p<0.01), GM-CSF (p<0.01), TET2 (p<0.01), HK1 (p<0.01), HK2 (p<0.01), and PFKFB3 (p<0.01). D-mannose also inhibited the recruitment and activation of neutrophils in LPS-treated rat gingival tissues. In vitro, the results of IF showed that D-mannose inhibited the activation of neutrophils stimulated by LPS, downregulated the expression of IL-1β (p < 0.05), IL-6, IL-17 (p < 0.01), and TET2 (p < 0.01), and upregulated the expression of IL-10 (p < 0.01).ConclusionsD-mannose can alleviate chronic periodontitis in rats by regulating the functions of neutrophils, potentially associated with the expression of TET2 and glycolysis, providing new insights into the potential application of D-mannose to chronic periodontitis.

Exosome-like nanovesicle targeting visceral adipose tissue inflammation mitigates heart failure with preserved ejection fraction

Abstract Introduction Heart failure (HF) with preserved ejection fraction (HFpEF) is associated with chronic systemic inflammation and obesity, with limited therapies. Inflammation in visceral adipose tissue (VAT) was significantly evoked in obese mice; while mesenchymal stem cell (MSC), especially atorvastatin (ATV)-pretreated MSC, could promote anti-inflammatory phenotype switch of immunocytes (e.g. macrophages), serving as a promising therapy for obesity-related HFpEF. Thus, the study was designed to prepare a novel ATV-pretreated MSC-derived exosome-like nanovesicles (ELNVs) and assess the anti-inflammatory and cardioprotective effects of ELNVs. Methods ELNVs derived from unpretreated MSC (ELNVM) or ATV-pretreated MSC (ELNVMA) were prepared and characterized (Figure 1). PKH-26-labelled ELNVs were incubated with THP1 cells for cell uptake assay. For anti-inflammatory studies in vitro, ELNVs (5×1010/mL) were added into the medium, followed by the induction of M1 or M2 polarization. The expressions of iNOS and Arg1 were measured by immunofluorescence (IF) staining. A 2-hit preclinical mice model of HFpEF, established by a 12-week high-fat diet (HFD) and Nω-Nitro-L-arginine methyl ester hydrochloride administration (0.5 g/L), was applied and received normal saline (NS) or ELNVs (i.p., 5×1011/kg body weight, 3 times a week). Functional and histological analysis including echocardiography, exhaustion test, WGA staining for hearts and western blot for VAT were performed. Quantitative polymerase chain reaction (qPCR) of key long non-coding RNAs (lncRNAs) was used to identify the mediator in ELNVs. Results The mean diameters of ELNVM and ELNVMA were 109.5 and 114.6 nm respectively and both ELNV types could be taken by THP1 cells. VAT inflammation was activated in HFpEF mice compared to the control in terms of the protein levels of iNOS and Arg1. An elevated expression of Arg1 and a decreased level of iNOS was observed in THP1 cells in vitro and VAT in vivo in both ELNV groups (P&amp;lt;0.05, Figure 1 and 2). Consistently, levels of interleukin (IL)-1β were increased in VAT of HFpEF mice, which were downregulated in ELNVM and ELNVMA groups. Both ELNVs could reduce the E/E’, and attenuate cardiac hypertrophy (heart weight to tibial length) and pulmonary congestion (lung weight [wet to dry]) (all P&amp;lt;0.05), with more pronounced improvements in ELNVMA group (Figure 2). Results of qPCR indicated three lncRNA candidates (TARID, H19 and KLF3-AS1) that were previously reported to inhibit M1 polarization or promote M2 polarization, were elevated in ELNVMA compared to ELNVM (P&amp;lt;0.05). Conclusion VAT inflammation was evoked in HFpEF mice and contributed to the development of HFpEF. This pilot study developed a novel ATV-pretreated ELNVs, which might serve as a promising tool for alleviating HFpEF due to the charming anti-inflammatory and cardioprotective effects mediated by regulating VAT macrophages.Figure 1.ELNVs’ PreparationFigure 2.Anti-inflammation of ELNVs

Establishment and analysis of an animal model for nutcracker syndrome.

This study investigated the effects of renal vein congestion of varying durations on kidney damage by establishing an animal model of Nutcracker Syndrome (NCS). In addition, we assessed whether renal damage improved after relieving the congestion. We established a rat model of NCS and relieved renal congestion through secondary surgery, with renal vein reperfusion confirmed by animal ultrasound. Histopathological changes in congested kidneys were evaluated at 3, 6, and 9days using HE and Masson staining, and the expression of renal injury factors was determined using q-PCR. Renal tissue pathology changes were evident with prolonged obstruction time. Compared to the sham-operated group, no apparent fibrosis was observed in the kidneys at 3days post-congestion, whereas fibrosis was most severe at 9days post-congestion, with no significant improvement observed even after blood flow restoration. Six days post-congestion relief, some alleviation of renal fibrosis occurred. q-PCR results indicated a significant reduction in the expression of fibrosis markers after 3 or 6days of renal vein ligation release. Following blood flow restoration on the 9th day post-congestion, no significant changes in fibrosis markers were noted. Prolonged renal vein congestion in a rat model of NCS resulted in severe kidney damage and significant fibrosis after 9days. While relieving congestion within 6days led to some improvement, no reduction in fibrosis occurred after 9days, underscoring the importance of congestion duration in renal pathology.

Study of the Role of E2F1 and TMEM132A in Prostate Cancer Development.

Identify transcription factors and target genes associated with prostate cancer, offering new therapy approaches. Gene Set Enrichment Analysis (GSEA) investigates early 2 factor (E2F) transcription factor family roles in prostate cancer using the TCGA database. Survival analysis examined E2F factors and patient survival connections. Dataset analysis identified E2F1-involved key genes. Quantitative Real-time PCR (qPCR), which combines ultrasound-guided methods to collect clinical samples from prostate cancer patients, was utilized to determine the expression levels of E2F1 and its target genes in patient samples and cancer cells. The effect of E2F1 and its target gene expression alterations on prostate cell proliferation was examined utilizing the cell counting kit-8 (CCK8) technique. Double fluorescence enzyme experiment verified E2F1-target gene connections. E2F family genes induce prostate cancer and show correlated co-expression. E2F1, E2F2, E2F3, E2F5, and E2F7 were considerably over-expressed in prostate cancer tissues. While E2F4 and E2F6 were notably underexpressed, there was no statistically important change in the E2F8 expression between prostate cancer and surrounding tissues. High expression of E2F genes is associated with lower patient survival. The transmemrane protein 132 (TMEM132A) was identified as a key gene for E2F1 action and is associated with poor prognosis in patients. The essential gene for E2F1 function, TMEM132A, was discovered. According to the qPCR results, E2F1 and TMEM132A are considerably expressed in cancer cells and patient samples. Interfering with its expression significantly inhibited the proliferation ability of cancer cells. The double luciferase experiment showed that E2F1 regulates the expression level in phase by binding directly to the TMEM132A promoter. The E2F transcription factor family induces prostate cancer and correlates with poor prognosis. E2F1 directly regulates TMEM132A by binding its promoter and controlling the degree of protein expression, thereby affecting cancer cell growth.

MBene Nanosheets with DNA Adsorbability for Circulating Tumor DNA Assay via Fluorescence Biosensing and Paper‐Based Microfluidic POCT

AbstractThe detection of circulating tumor DNA (ctDNA) in blood is significant for non‐invasive cancer diagnosis and treatment monitoring. Herein, MBene nanosheets is synthesized and compared with MXene via Density Functional Theory (DFT) calculations and fluorescence kinetic evaluations, for first time, revealing MBene's exceptional DNA adsorbability and discrimination to single‐stranded DNA (ssDNA) and double‐stranded DNA (dsDNA). Then, a sensitive fluorescence biosensor for ctDNA detection is developed, demonstrating impressive performance. To facilitate point‐of‐care testing (POCT) ctDNA, a paper‐based microfluidic chip incorporated a delay area and a mixing channel is designed. The constricted‐expanded structure channel optimization is guided by numerical simulations and experiments. A WeChat mini program named “ctDNA Detection” is designed for readout assay. Furthermore, cell and mice serum samples are analyzed, with Magnetic Bead@Graphene Oxide (MB@GO) and clutch probes for magnetic pre‐enrichment. The results accuracy is confirmed by its consistency with standard qPCR results (AUC = 1). The successful detection of ctDNA in post‐surgery mouse models underscored the biosensor's potential for cancer treatment monitoring. Thus, this research not only advanced the understanding of the MBene‐DNA interaction in biosensing, but also can pave the way for novel applications in bioimaging and nanopore‐based nucleic acid sequencing, leveraging the digital transformation of DNA base‐MBene adsorption differences.

Genetic dissection of Meloidogyne incognita resistance genes based on VIGS functional analysis in Cucumis metuliferus

The southern root-knot nematode, Meloidogyne incognita, is a highly serious plant parasitic nematode species that causes significant economic losses in various crops, including cucumber (Cucumis sativus L.). Currently, there are no commercial cultivars available with resistance to M. incognita in cucumber. However, the African horned melon (Cucumis metuliferus Naud.), a semi-wild relative of cucumber, has shown high resistance to M. incognita. In this study, we constructed an ultrahigh-density genetic linkage bin-map using low-coverage sequences from an F2 population generated through the cross between C. metuliferus inbred lines CM3 and CM27. Finally, we identified a QTL (quantitative trait locus, QTL3.1) with a LOD (logarithm of the odds) score of 3.84, explaining 8.4% of the resistance variation. Subsequently, by combining the results of qPCR (quantitative PCR) and VIGS (virus-induced gene silencing), we identified two genes, EVM0025394 and EVM0006042, that are potentially involved in the resistance to M. incognita in CM3. The identification of QTLs and candidate genes in this study serve as a basis for further functional analysis and lay the groundwork for harnessing this resistance trait.

Non-targeted metabolomics and pseudo-targeted lipidomics combined with gut microbes reveal the protective effects of Causonis japonica (Thunb.) Raf. in ulcerative colitis mice.

Ulcerative colitis (UC) is an inflammatory bowel disease characterized by recurrent inflammatory tissue damage to the intestinal mucosa and forming intestinal epithelial ulcers. It is one of the most intractable diseases in the world. To date, the mechanism is unclear. Causonis japonica (Thunb.) Raf. (Wu Lianmei in Chinese; WLM), a traditional Chinese medicine, which has a long history as an anti-inflammatory, but its effect on UC was unconfirmed yet. Therefore, we established a dextran sodium sulfate (DSS)-induced UC mice model and evaluated the therapeutic effect of WLM extract. The results indicated that WLM inhibits DSS-induced inflammatory response in colitis in vivo, decrease DSS-induced clinical manifestations, reverses colon length shortening, and reduces tissue damage. The results of ELISA kits suggested that WLM could reverse the levels of DSS-induced inflammatory factors. To explore the mechanism of WLM in treating DSS-induced UC, 1H NMR and UHPLC-Q/Orbitrap MS were used to perform non-targeted metabolomics analysis; 21 differential metabolites in colon tissues were closely related to UC. Meanwhile, the pseudo-targeted lipidomics based on UHPLC-Q/Trap MS was used to analyze lipid metabolism disorders, and 60 differential lipid compounds were screened. These differential compounds were mainly involved in glycerophospholipid, arachidonic acid, glycerolipid, citric acid, tyrosine, and ether lipid metabolisms. The analysis of gut microbial showed that WLM may improve the symptoms of UC mice by reducing the abundance of Helicobacter and Streptococcus and increasing the abundance of Limosilactobacillus and Akkermansia. Moreover, the real-time qPCR results showed that WLM extract could decrease the mRNA levels of inflammatory factors and may be associated with protecting the integrity of intestinal mucosal barrier by destroying in vivo metabolic pathways, especially by regulating energy and lipid metabolisms and reducing inflammatory reactions. It provides a beneficial reference for studying WLM to elucidate the therapeutic mechanism of UC.

Identification, expression, and function analysis of Rbpms2 splicing variants in Japanese flounder gonad

Rbpms2, an RNA-binding protein with multiple splicing (Rbpms), can interact with RNAs to involve oocyte development, thereby influencing female sex differentiation in vertebrates. Here, two splicing variants of the Rbpms2 gene from Japanese flounder (Paralichthys olivaceus) were identified, namely Rbpms2.1 and Rbpms2.2. The two variants exhibited 98.22 % amino acid homology, both featuring an RNA recognition motif (RRM) domain spanning positions 98–170 amino acids. They were relatively conserved throughout phylogenetic evolution. Differently, the C-terminal region of the Rbpms2.1 contains five additional sequential amino acids (–VRDQP–) compared to Rbpms2.2. The real-time qPCR results demonstrated that Rbpms2.1 and Rbpms2.2 had relatively abundant expression in the gonads of adult Japanese flounder, with higher expression levels in the ovary compared to the testis (P < 0.05). In situ hybridization results showed strong positive expression of Rbpms2 mRNA in oocytes at stages I-III during the V stage of ovarian development. In the testis atstage IV, the expression of Rbpms2 mRNA was mainly concentrated on primary spermatocytes. Importantly, Rbpms2 binding sites were found in the 3′UTR, 5′UTR, and ORF regions of the sex-related genes including dmrt1, sox9, amh, foxl2, and wnt4. siRNA interference and overexpression analysis of Rbpms2.1 and Rbpms2.2 in primary cells of the ovary and testis showed that Rbpms2 can repress the expression of male-related genes (dmrt1, sox9, and amh) and significantly promote the expression of female-related genes (foxl2 and wnt4). Our results revealed that Rbpms2 may play a critical role by targeting the sex-related genes in the gonad development of Japanese flounder.

A Novel Trichinella spiralis Galectin Strengthens the Macrophage ADCC Killing of Larvae via Driving M1 Polarization.

Galectin recognizes β-galactosides through its carbohydrate recognition domains (CRDs). This study aimed to determine the biological features of a novel Trichinella spiralis galectin (galactoside-binding lectin family protein, TsGLFP) and its role in driving macrophage M1 polarization and enhancing ADCC killing of larvae. TsGLFP belongs to the galectin family and has two CRDs. The complete TsGLFP cDNA sequence was cloned and then expressed in Escherichia coli BL21. The results of qPCR, Western blot, and indirect immunofluorescence tests (IIFTs) revealed that TsGLFP was expressed in various stages of T. spiralis worms and principally localized at the cuticle and around the female embryos of the nematode. rTsGLFP had the function of agglutinating mouse erythrocytes, and this agglutination activity could be inhibited by lactose. After the mouse macrophage RAW264.7 was incubated with rTsGLFP, the expression level of the M1 genes (iNOS, IL-6, and TNF-α) and NO production were obviously increased. After incubating macrophages with rTsGLFP, there was a noticeable rise in the expression levels of p-IκB-α and p-NF-κB p65. Additionally, rTsGLFP enhanced the macrophage's ability to kill newborn larvae by ADCC cytotoxicity. When the macrophages were pretreated with the specific p-NF-κB p65 inhibitor PDTC, and then stimulated with rTsGLFP, the expression levels of iNOS, NO, and p-NF-κB p65 and the macrophages' ADCC cytotoxicity were distinctly decreased. These findings indicated that rTsGLFP enhanced the macrophage ADCC killing of larvae by driving M1 polarization through activating the NF-κB pathway.

Molecular Actions of Enicostemma hyssopifolium Whole Plant Extract on HPV18-Infected Human Cervical Cancer (HeLa) Cells.

Enicostemma hyssopifolium (E. hyssopifolium) contains several bioactive compounds with anti-cancer activities. This study was performed to investigate the molecular effects of E. hyssopifolium on HPV18-containing HeLa cells. The methanol extract of E. hyssopifolium whole plant was tested for cytotoxicity by MTT assay. A lower and higher dose (80 and 160 μg/mL) to IC50 were analyzed for colonization inhibition (Clonogenic assay), cell cycle arrest (FACS analysis), and induction of apoptosis (AO/EtBr staining fluorescent microscopy and FACS analysis) and DNA fragmentation (comet assay). The HPV 18 E6 gene expression in treated cells was analyzed using RT-PCR and qPCR. A significant dose-dependent anti-proliferative activity (IC50 - 108.25±2 μg/mL) and inhibition of colony formation cell line were observed using both treatments. Treatment with 80 μg/mL of extract was found to result in a higher percent of cell cycle arrest at G0/G1 and G2M phases with more early apoptosis, while 160 μg/mL resulted in more cell cycle arrest at SUBG0 and S phases with late apoptosis for control. The comet assay also demonstrated a highly significant increase in DNA fragmentation after treatment with 160 μg/mL of extract (tail moments-19.536 ± 17.8), while 80 μg/mL of extract treatment showed non-significant tail moment (8.152 ± 13.0) compared to control (8.038 ± 12.0). The RT-PCR and qPCR results showed a significant reduction in the expression of the HPV18 E6 gene in HeLa cells treated with 160 μg/mL of extract, while 80 μg/mL did not show a significant reduction. The 160 μg/mL methanol extract of E. hyssopifolium demonstrated highly significant anti-cancer molecular effects in HeLa cells.

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

Quantitative real time PCR for distinction between Pneumocystis jirovecii infection/colonization in hospitalized patients.

Identification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency. To set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)-rRNA gene. Based on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probe-based qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: ≤32 as infection, >35 as colonization, and a grey zone between these values (32 < X ≤ 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR). This study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis-related conditions.

Comparison of multi-parallel quantitative real-time PCRs targeting different DNA regions and detecting soil-transmitted helminths in stool

BackgroundSoil-transmitted helminths infect an estimated 18% of the world’s population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets.MethodsWe aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences.ResultsFor Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28–0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement.ConclusionsThere is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.Graphical abstract