Quantitative PCR Assay Research Articles

Spatiotemporal dynamics of Phytophthora infestans airborne inoculum in Belgium

AbstractFor nearly a century, scientists have strived to model the development of Phytophthora infestans (Pi) to predict late blight infections in potatoes. This has led to the use of decision support systems (DSSs) that rely on forecasting models based on environmental parameters. All these models assume that the primary inoculum is ubiquitous. This study focuses on the spatiotemporal distribution of airborne inoculum of Pi to assess the value of incorporating quantitative data of this variable in improving the prediction of primary infection of potato late blight in fields. The daily spatiotemporal distribution of Pi airborne inoculum was studied from 2019 to 2022 at Ath, Gembloux, Libramont and Louvain-la-Neuve in Belgium by combining Burkard spore traps with a quantitative PCR assays. The quantities of Pi inoculum trapped daily ranged from 0 to 4903 (expressed as sporangia equivalent) depending on the site and the year. The appearance of late blight symptoms in untreated plots located close to spore traps was assessed shortly after the detection of airborne inoculum in all monitored sites. A comparison between airborne inoculum detection and the recommendations provided by three DSSs revealed that fungicide treatments are often recommended at the beginning of the season when airborne inoculum is absent. Two field trials performed in 2021 and 2022 showed that the number of fungicide treatments could be reduced by considering the presence of the inoculum, particularly by delaying the first application. Further knowledge of the relationships between disease pressure, airborne inoculum and meteorological conditions will provide valuable information for improving DSSs and reducing fungicide use.

MicroRNA Temporal-Specific Expression Profiles Reveal longissimus dorsi Muscle Development in Tianzhu White Yak.

As a class of regulatory factors, microRNAs (miRNAs) play an important role in regulating normal muscle development and fat deposition. Muscle and adipose tissues, as major components of the animal organism, are also economically important traits in livestock production. However, the effect of miRNA expression profiles on the development of muscle and adipose tissues in yak is currently unknown. In this study, we performed RNA sequencing (RNA-Seq) on Tianzhu white yak longissimus dorsi muscle tissue obtained from calves (6 months of age, M6, n = 6) and young (30 months of age, M30, n = 6) and adult yak (54 months of age, M54, n = 6) to identify which miRNAs are differentially expressed and to investigate their temporal expression profiles, establishing a regulatory network of miRNAs associated with the development of muscle and adipose. The results showed that 1191 miRNAs and 22061 mRNAs were screened across the three stages, of which the numbers of differentially expressed miRNAs (DE miRNAs) and differentially expressed mRNAs (DE mRNAs) were 225 and 450, respectively. The expression levels of the nine DE miRNAs were confirmed using a reverse transcription quantitative PCR (RT-qPCR) assay, and the trend of the assay results was generally consistent with the trend of the transcriptome profiles. Based on the expression trend, DE miRNAs were categorized into eight different expression patterns. Regarding the expression of DE miRNAs in sub-trends Profile 1 and Profile 2 (p < 0.05), the gene expression patterns were upregulated (87 DE miRNAs). Gene ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG) analyses showed that the identified DE miRNAs and DE mRNAs were enriched in pathway entries associated with muscle and intramuscular fat (IMF) growth and development. On this basis, we constructed a DE miRNA-mRNA interaction network. We found that some DE mRNAs of interest overlapped with miRNA target genes, such as ACSL3, FOXO3, FBXO30, FGFBP4, TSKU, MYH10 (muscle development), ACOX1, FADS2, EIF4E2, SCD1, EL0VL5, and ACACB (intramuscular fat deposition). These results provide a valuable resource for further studies on the molecular mechanisms of muscle tissue development in yak and also lay a foundation for investigating the interactions between genes and miRNAs.

Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya

Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya

Ensemble for evaluating diagnostic efficacy of non-invasive indices in predicting liver fibrosis in untreated hepatitis C virus population.

Hepatitis C virus (HCV) infection progresses through various phases, starting with inflammation and ending with hepatocellular carcinoma. There are several invasive and non-invasive methods to diagnose chronic HCV infection. The invasive methods have their benefits but are linked to morbidity and complications. Thus, it is important to analyze the potential of non-invasive methods as an alternative. Shear wave elastography (SWE) is a non-invasive imaging tool widely validated in clinical and research studies as a surrogate marker of liver fibrosis. Liver fibrosis determination by invasive liver biopsy and non-invasive SWE agree closely in clinical studies and therefore both are gold standards. To analyzed the diagnostic efficacy of non-invasive indices [serum fibronectin, aspartate aminotransferase to platelet ratio index (APRI), alanine aminotransferase ratio (AAR), and fibrosis-4 (FIB-4)] in relation to SWE. We have used an Artificial Intelligence method to predict the severity of liver fibrosis and uncover the complex relationship between non-invasive indices and fibrosis severity. We have conducted a hospital-based study considering 100 untreated patients detected as HCV positive using a quantitative Real-Time Polymerase Chain Reaction assay. We performed statistical and probabilistic analyses to determine the relationship between non-invasive indices and the severity of fibrosis. We also used standard diagnostic methods to measure the diagnostic accuracy for all the subjects. The results of our study showed that fibronectin is a highly accurate diagnostic tool for predicting fibrosis stages (mild, moderate, and severe). This was based on its sensitivity (100%, 92.2%, 96.2%), specificity (96%, 100%, 98.6%), Youden's index (0.960, 0.922, 0.948), area under receiver operating characteristic curve (0.999, 0.993, 0.922), and Likelihood test (LR+ > 10 and LR- < 0.1). Additionally, our Bayesian Network analysis revealed that fibronectin (> 200), AAR (> 1), APRI (> 3), and FIB-4 (> 4) were all strongly associated with patients who had severe fibrosis, with a 100% probability. We have found a strong correlation between fibronectin and liver fibrosis progression in HCV patients. Additionally, we observed that the severity of liver fibrosis increases with an increase in the non-invasive indices that we investigated.

Bionomics and distribution of malaria vectors in Kisumu city, Western Kenya: Implications for urban malaria transmission.

Increasing urbanization in tropical Africa may create new niches for malaria vectors, potentially leading to higher disease transmission rates. Vector control efforts remain largely targeted at ecologically rural bio-complexities with multiple hosts. Understanding mosquito species composition, ecology, host diversity and biting behavior in urban areas is crucial for planning effective control. This study assessed mosquito species diversity, abundance, behavioral patterns, and Plasmodium sporozoite infection rates of Anopheles vectors along an urban-rural transect in Kisumu city, western Kenya. Indoor and outdoor host-seeking and resting adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and mechanical aspirators (Prokopack) along an urban-rural transect. Females Anopheles mosquitoes collected were identified using morphological taxonomic keys to species level. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further processed using polymerase chain reaction (PCR) to identify members of each complex/group. Subsequently, sporozoite infection rates of the anopheline mosquitoes were determined using a multiplexed real-time quantitative PCR (qPCR) assay. A total of 3,394 female Anopheles mosquitoes were collected and identified. These comprised of An. gambiae s.l. (68%), An. funestus group (19.8%), An. coustani (7.8%), An. pharoensis (2.6%), An. maculipalipis (1.6%), and An. leesoni(0.2%). All six species were found in urban zone, but only three were found in peri-urban and rural sites. Overall, urban collections accounted for the majority of these collections (55.5%) of mosquitoes collected, followed by those from peri-urban (30%) and rural sites (14.5%). Species distribution across the three ecotypes showed Anopheles arabiensis was the dominant species in urban (84.3%) and peri-urban (89%) sites, while An. gambiae s.s. was predominantly found in the rural zone (60.2%) alongside An. arabiensis (39.7%). Anopheles funestus was the predominant species in peri-urban (98.4%) and rural (85.7%) areas, with An. leesoni accounted for 1.6% and 14.3%, respectively. In urban areas, all samples from the An. funestus group were identified as An. funestus s.s.. Majority (55.5%) of Anopheles mosquitoes were collected indoors, while secondary vectors were primarily caught outdoors. Overall, sporozoite rates were higher outdoors 3.5% compared to indoors 1.45% in rural areas. Specifically, sporozoite infectivity rates for An. funestus, An. gambiae s.s and An. arabiensis collected indoors in the rural zone was 2.5%, 1.4% and 1% respectively. Outdoors in rural areas, An. gambiae had a sporozoite rate of 5.3%, while An. arabiensis had a rate of 2.1%. In peri-urban areas An. gambiae had a sporozoite rate of 2.3%. No sporozoites were detected in samples from urban sites. The study highlights a shift of diversity of Anopheles species towards urban areas with increased outdoor activity, and significant outdoor malaria transmission in rural and peri-urban areas, emphasizing the need for tools targeting outdoor-biting mosquitoes. The presence of An. funestus in urban settings is of interest and highlights the critical importance of sustained entomological surveillance to inform integrated vector control and prevent future transmission risks.

Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii

This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.

A quantitative real-time PCR assay for rapid detection and quantification of Amyloodinium ocellatum parasites in seawater samples

Amyloodinium ocellatum is one of the most important ectoparasites of marine fish, causing amyloodiniosis and mass mortality in aquaculture. To date, no diagnostic method has been developed to detect and quantify parasite abundance in seawater. In this study, a quantitative real-time PCR (qPCR) assay using the ITS-F3 and ITS-R3 primer pair based on the internal transcribed spacers of nuclear ribosomal DNA (ITS rDNA) of A. ocellatum was successfully established for the detection and quantification of dinospores in seawater. The dinospores were collected by suction filtration of seawater using a vacuum pump, and examination by light microscopy and scanning electron microscopy (SEM) showed that the dinospores were adsorbed on the surface of the cellulose acetate membrane. The linear relationship between seawater parasite abundance and Ct values followed the model y = −3.0607 x + 29.919 with a coefficient of determination (R2) of 0.985. The results of the assay showed high reproducibility and no cross-reactivity with other aquatic pathogens. The assay successfully detected dinospores (100 % success rate) at concentrations of 8 and 10 cell per 300 mL of seawater. The protective fluid buffer effectively protected the dinospores DNA from degradation for over 96 h. The method was used to investigate the life span of dinospores in seawater and the incubation patterns of tomonts in the tank, the results showed that a small fraction of dinospores can survive for more than 7 days in seawater and the process of dinospores incubation from the tomonts persisted up to 84 h. This study provides a valuable quantitative diagnostic tool to assist in the early monitoring and understanding of disease progression in amyloodinosis.

Proteomic profiling of oleamide-mediated polarization in a primary human monocyte-derived tumor-associated macrophages (TAMs) model: a functional analysis.

Tumor-associated macrophages (TAMs) play a critical function in the development of tumors and are associated with protumor M2 phenotypes. Shifting TAMs towards antitumor M1 phenotypes holds promise for tumor immunotherapy. Oleamide, a primary fatty acid amide, has emerged as a potent anticancer and immunomodulatory compound. However, the regulatory effects of oleamide on TAM phenotypes remain unclear. We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to study the influence of oleamide on primary human monocyte-derived TAM phenotypes, and we investigated the protein expression profiles based on mass spectrometry to analyze the effect of oleamide on macrophage polarization. Moreover, the advantageous binding scores between oleamide and these target candidate proteins are examined using molecular docking. Our study revealed that oleamide effectively suppressed the M2-like TAM phenotype by reducing interleukin (IL)-10 production and downregulating M2-like markers, including vascular endothelial growth factor A (VEGFA), MYC proto-oncogene, bHLH transcription factor (c-Myc), and mannose receptor C-type 1 (CD206). Moreover, the conditioned medium derived from oleamide-treated TAMs induces apoptosis of MDA-MB-231 breast cancer cells. Proteomic analysis identified 20 candidate up- and down-regulation proteins targeted by oleamide, showing modulation activity associated with the promotion of the M1-like phenotype. Furthermore, molecular docking demonstrated favorable binding scores between oleamide and these candidate proteins. Collectively, our findings suggest that oleamide exerts a potent antitumor effect by promoting the antitumor M1-like TAM phenotype. These novel insights provide valuable resources for further investigations into oleamide and macrophage polarization which inhibit the progression of breast cancer, which may provide insight into immunotherapeutic approaches for cancer.

Estrogen, via ESR2 receptor, prevents oxidative stress-induced Müller cell death and stimulates FGF2 production independently of NRF2, attenuating retinal degeneration

In this study, we aimed to investigate the effects of the deficient antioxidative gene, nuclear factor-erythroid 2-related factor 2 (Nrf2), on 17β-estradiol (E2)-mediated oxidative stress response, with a specific focus on growth factor production and cell death in Müller cells and retinal tissue. Administration of hydrogen peroxide (H2O2) reduced the viability of Müller cells derived from Nrf2 wild-type (WT) and knockout (KO) mice. However, this effect was more significant in the KO cells than in the WT cells. Pretreatment with E2 inhibited H2O2-induced cell death in both Nrf2 WT and KO Müller cell genotypes. Small interfering RNA-mediated gene silencing of estrogen receptor 2 (Esr2) attenuated the cell survival-promoting activity of E2 in Nrf2 KO Müller cells, while other identified estrogen receptors, Esr1 or G protein-coupled estrogen receptor 1 (Gper1), had no effect. Western blotting revealed higher ESR2 expression levels in Nrf2 KO cells than in WT Müller cells. Conditioned media from E2-and H2O2-treated Nrf2 WT or KO Müller cells enhanced the dissociated retinal cell viability compared with H2O2-treated cells. Both quantitative reverse-transcription polymerase chain reaction assay (qRT-PCR) and enzyme-linked immunosorbent assay exhibited a significant increase in fibroblast growth factor 2 (FGF2) expression levels in E2-and H2O2-treated Nrf2 WT and KO Müller cells compared to those in E2-treated cells. In vivo, administration of N-methyl-N-nitrosourea (MNU) reduced the thickness and cell density of the outer nuclear layer (ONL) in Nrf2 KO mice and enhanced the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the ONL. However, E2 administration attenuated these defects in MNU-treated mice. Concomitant administration of MNU and E2 enhanced FGF2 protein levels in retinal lysates of Nrf2 KO mice. In conclusion, E2 demonstrated a significant role in preventing oxidative stress-induced retinal cell death by stimulating FGF2 production in Müller cells, independent of the Nrf2 gene. Based on these findings, we anticipate that exogenous administration of estrogens or ESR2-selective agonists could aid in treating patients with oxidative stress-related retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

Neurotoxic mechanisms of dexamethasone in SH-SY5Y neuroblastoma cells: Insights into bioenergetics, oxidative stress, and apoptosis

Despite the known therapeutic uses of dexamethasone (DEX), the specific mechanisms underlying its neurotoxic effects in neuronal cells, particularly in undifferentiated human neuroblastoma (SH-SY5Y) cells, remain inadequately understood. This study aims to elucidate these mechanisms, emphasizing bioenergetics, oxidative stress, and apoptosis, thereby providing novel insights into the cellular vulnerabilities induced by chronic DEX exposure. The findings revealed significant reductions in cell viability, altered membrane integrity with LDH leakage, decreased intracellular ATP production, and the electron transport chain complexes I and III activity inhibition. DEX significantly increased the release of the reactive species and peroxidation of lipids, as well as of Nrf2 expression. At the same time, it simultaneously led to a decline in the activities of the antioxidant catalase and superoxide dismutase enzymes, along with a depletion of glutathione reserves. The apoptosis process was exhibited by a significant elevation of caspases 3 and 8 activities with overexpression of mRNA BAX, inhibition of BCL-2, and a significant upregulation of the BAX/BCL-2 ratio. Assessment of neuronal development genes (GAP43, CAMK2A, CAMK2B, TUBB3, and Wnts) by quantitative PCR assay showed increased expression of CAMK2A, CAMK2B, and Wnt3a with a significant reduction in GAP43 mRNA levels. Collectively, this study proved that DEX was cytotoxic to SH-SY5Y via bioenergetic disruption, mitochondrial dysfunction, oxidative stress, and apoptosis.

Congenital CMV infection - what we know about the symptoms, treatment and prevention? - Review of literature

Introduction: Cytomegalovirus (CMV) is a double-strand DNA virus, member of the viral family known as Herpesviridae which is wide-spread. CMV infection is the most common congenital infection worldwide. Disease is mostly asymptomatic for healthy people, therefore is detected late, when the children start presenting symptoms. Unfortunately the congenital CMV infection leads to the serious health problems for infants. Detection of the virus is typically done through quantitative PCR assays. Infection especially affects nervous system and can present infant’s deafness. Earlier it was equivalent with the children being deaf till the end of life, but nowadays thanks to the development of laryngological methods (ex. cochlear implantation) patients have chance to grow up correctly. Material and methods: A literature search was conducted using Google Scholar, PubMed, and Web of Science databases. We searched articles by entering key words in the appropriate configuration: “CMV”, “Congenital CMV infection”, “valganciclovir”, “infant’s deafness”. Scientific articles published between 1991–2023 were analyzed from all over the world and guidelines of Polish Society of Epidemiology and Infectious Diseases.Conclusions: Currently we haven’t got prevention towards congenital CMV, only the treatment for infants: valganciclovir. It stops development of severe symptoms. There are available analysis, where using valganciclovir during pregnancy reduces the possibility or soften the course of the congenital CMV infection , but it’s not the obligatory guidelines. In many countries researchers want to conduct screening test for CMV IgG among the pregnant women.

Bone marrow-derived mesenchymal stem cell-derived exosome-loaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage

BACKGROUND Diabetic intracerebral hemorrhage (ICH) is a serious complication of diabetes. The role and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes (BMSC-exo) in neuroinflammation post-ICH in patients with diabetes are unknown. In this study, we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation. AIM To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage. METHODS BMSC-exo were isolated from mouse BMSC media. This was followed by transfection with microRNA-129-5p (miR-129-5p). BMSC-exo or miR-129-5p-overexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucose-affected BV2 cells for in vitro analyses. The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1 (HMGB1). Quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors, such as HMGB1, interleukin 6, interleukin 1β, toll-like receptor 4, and tumor necrosis factor α. Brain water content, neural function deficit score, and Evans blue were used to measure the neural function of mice. RESULTS Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery. MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation. Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases. Furthermore, we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA. CONCLUSION We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes, thereby improving the neurological function of the brain.

Bone marrow-derived mesenchymal stem cell-derived exosome-loaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage.

Diabetic intracerebral hemorrhage (ICH) is a serious complication of diabetes. The role and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes (BMSC-exo) in neuroinflammation post-ICH in patients with diabetes are unknown. In this study, we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation. To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage. BMSC-exo were isolated from mouse BMSC media. This was followed by transfection with microRNA-129-5p (miR-129-5p). BMSC-exo or miR-129-5p-overexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucose-affected BV2 cells for in vitro analyses. The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1 (HMGB1). Quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors, such as HMGB1, interleukin 6, interleukin 1β, toll-like receptor 4, and tumor necrosis factor α. Brain water content, neural function deficit score, and Evans blue were used to measure the neural function of mice. Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery. MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation. Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases. Furthermore, we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA. We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes, thereby improving the neurological function of the brain.

Abstract B089: Identification of novel drivers of gemcitabine resistance in pancreatic cancer

Abstract Pancreatic cancer is an aggressive disease with limited therapeutic options. Gemcitabine is a clinically used chemotherapy for pancreatic cancer treatment but has limited efficacy. The molecular mechanisms responsible for gemcitabine resistance in pancreatic cancer are still unclear. Here, we analyzed the genomics of drug sensitivity in cancer dataset and identified various pancreatic cancer cells with distinct sensitivity to gemcitabine. Validation with proliferation and clonogenic assays confirmed that while CFPAC1 and PANC0327 are highly gemcitabine-sensitive, ASPC1 and PANC1 display intrinsic gemcitabine resistance. Analysis of the gene expression profile of these cells identified several genes that are differentially upregulated in resistant versus sensitive cells. Pathway enrichment analysis using the differential genes revealed the upregulation of Hippo, Wnt, MAPK signaling pathways as well as autophagy and glutathione metabolism in the resistant cells. Using quantitative PCR, immunoblotting and biochemical assays, and extracellular flux analysis methods, we have characterized the molecular distinctions between gemcitabine sensitive and resistant cells. Our results provide insight on the molecular mechanisms driving gemcitabine resistance in pancreatic cancer and could lead to novel therapeutic strategies to overcome resistance and improve patients’ treatment. Citation Format: Amina D Bilalbegovic, Bowen Fu, Zeribe Nwosu. Identification of novel drivers of gemcitabine resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B089.

Resting Cysts of the Toxic Dinoflagellate Gymnodinium catenatum (Dinophyceae) Ubiquitously Distribute along the Entire Coast of China, with Higher Abundance in Bloom-Prone Areas

Blooms of Gymnodinium catenatum have occurred occasionally in different areas of China and caused tremendous economic loss and even threatened human health. Not only is G. catenatum an important harmful-algal-bloom (HAB)-causing species, but also the only gymnodinioid dinoflagellate known to produce paralytic shellfish poisoning toxins (PSTs). Due to the germination of resting cysts, which often initiates blooms, the distribution and abundance of cysts in sediments and particularly the confirmation of cyst beds are important information for understanding and predicting dinoflagellate blooms. In this research, 199 sediment samples were collected from China’s coastal seas, ranging from the Beidaihe in the Bohai Sea (BS) to the southernmost sample from the Nansha Islands of the South China Sea (SCS). TaqMan quantitative PCR (qPCR) assays with species-specific primers and probes were developed to specifically detect the distribution and abundance of cysts in the 199 samples. The detection revealed that G. catenatum cysts were widely present in the sediments (126 of the 199 samples), with 93.55%, 74.65%, 42.37%, and 50% of the samples detected positively from the BS, YS, ECS and SCS, respectively, and covering the vast sea area from Nansha Islands to the Beidaihe area. The single-cyst morpho-molecular identification in the samples from Beidaihe confirmed the existence of G. catenatum cysts in the BS, and the positive detections of G. catenatum cysts using the qPCR methods. While G. catenatum cysts were widely distributed in all four seas of China, the average abundance was relatively low (1.0 cyst per gram of wet sediment). Three samples from the East China Sea (ECS), however, contained G. catenatum cysts at a relatively higher level (23 cysts g−1 wet sediment) than other sea areas, suggesting a pertinence of cyst abundance to the frequent occurrences of G. catenatum blooms in the area during recent years. Collectively, for G. catenatum being such an important toxic and HAB-causing species globally, the ubiquitous distribution of its cysts along the coastal waters of China and higher abundance in the bloom-prone areas warns us of a risk that cyst beds, although currently low in abundance, may seed HABs in any and many sea areas of China at any forthcoming year, and particularly those areas with records of frequent HABs outbreaks in the past.

Enhancement of Sesquiterpenoid Production by Methyl Jasmonate in Atractylodes chinensis Adventitious Root Culture and its Transcriptional Regulation.

The adventitious root (AR) culture of Atractylodes chinensis is an efficient platform for sustainable production of its sesquiterpenoid compounds (atractylon and β-eudesmol). However, their limited accumulation levels need an effective elicitation approach, and the present study solved this problem using methyl jasmonate (MeJA) as an elicitor. The effects of its treatment concentration and duration on metabolite production were investigated. The ARs treated with 100 µM MeJA for seven days increased atractylon and β-eudesmol by 3.64- and 1.90-fold, respectively, compared with the control. This study further performed transcriptome analysis to explore the transcriptional regulation mechanism of the MeJA elicitation. A total of 124,464 unigenes were identified in A. chinensis ARs, of which 3,568 genes were upregulated and 3,864 genes were downregulated under the MeJA treatment. The MeJA treatment activated the endogenous JA biosynthesis and signaling pathways and sesquiterpenoid biosynthesis. The MeJA treatment more significantly activated the MEP pathway than the MVA pathway. In addition, 14 genes encoding terpene synthase were identified to be significantly upregulated. A total of 2,700 transcription factors (TFs) were identified in A. chinensis ARs, of which Tify, MYB, and MADS were significantly enriched under the MeJA treatment. We predicted a new antagonistic interaction between MYC2 and CPP TFs, which was significantly regulated by the MeJA treatment. The results of real-time quantitative PCR and enzyme activity assays proved the reliability of the transcriptome data. This study will help improve the in vitro production system of A. chinensis sesquiterpenoids and understand the transcriptional regulation mechanism of MeJA elicitation.

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

Propagation of koi herpesvirus using embryonated chicken eggs: a potential substitute method for fish vaccine production?

Koi herpesvirus disease (KHVD) is caused by a large DNA virus that commonly infects carp, Cyprinus carpio (koi and common carp). KHVD has spread rapidly across the globe and caused high mortality in all ages of common carp and koi. Until now, no effective treatment has been applied to prevent KHV infection impacting the mass production of koi and common carp . An environmentally friendly alternative strategy for controlling fish disease is vaccination. One of the challenges facing conventional viral vaccine production is the requirement for koi or common carp cell cultures, which must be frequently maintained with expensive materials required for virus propagation. This study aims to obtain KHV that has been propagated in embryonated chicken eggs (ECE) to formulate an affordable KHV vaccine for the aquaculture industry. This research consisted of three stages; the first stage was virus inoculation into various parts of eggs (allantoic fluid, chorioallantoic membrane, amniotic fluid, and egg yolk). The second stage was observing viral growth and collecting ECE fluid and membranes. The third stage involved quantitatively determining the viral genomic copy numbers using the quantitative Polymerase Chain Reaction (qPCR) assay. KHV propagation in various parts of ECE resulted in varying viral genomic copy numbers with a high DNA copy number reported in allantoic fluid on the third day after inoculation. Further work is required to monitor virus titer in later passages and optimize methodology for using ECE as the potential alternative to cultured cells as the medium for virus propagation. In the future the system could be developed to produce promising vaccine candidates with more affordable vaccine prices for the Indonesian fish farming industry.

N6-Methyladenosine Modification-Related Genes Express Differentially in Sterile Male Cattle-Yaks.

N6-methyladenosine (m6A), an RNA post-transcriptional modification, plays a crucial role in spermatogenesis. Cattle-yaks are interspecific hybrid offsprings of yak and cattle, and male cattle-yaks are sterile. This study aims to investigate the role of m6A modification in male cattle-yak infertility. Herein, testicular tissues were analyzed via histological observations, immunohistochemical assays, reverse-transcription quantitative polymerase chain reaction, Western blotting, and immunofluorescence assays. The results revealed that male cattle-yaks presented smaller testes (5.933 ± 0.4885 cm vs. 7.150 ± 0.3937 cm), with only single cell layers in seminiferous tubules, and weakened signals of m6A regulators such as METTL14 (methyltransferase-like 14), ALKBH5 (alpha-ketoglutarate-dependent hydroxylase homolog 5), FTO (fat mass and obesity-associated protein), and YTHDF2 (YTH N6-methyladenosine RNA binding protein F2), both at the RNA and protein levels, compared with those of yaks. Altogether, these findings suggest that m6A modification may play a crucial role in male cattle-yak sterility, providing a basis for future studies.

The m6A writer KIAA1429 regulates photoaging progression via MFAP4-dependent collagen synthesis

BackgroundN6-Methyladenosine (m6A) methylation, a common form of RNA modification, play an important role in the pathogenesis of various diseases and in the ontogeny of organisms. Nevertheless, the precise function of m6A methylation in photoaging remains unknown.ObjectivesThis study aims to investigate the biological role and underlying mechanism of m6A methylation in photoaging.Methodsm6A dot blot, Real-time quantitative PCR (RT-qPCR), western blot and immunohistochemical (IHC) assays were employed to detect the m6A level and specific m6A methylase in ultraviolet ray (UVR)-induced photoaging tissue. The profile of m6A-tagged mRNA was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis. Finally, we investigated the regulatory mechanism of KIAA1429 by MeRIP-qPCR, RNA knockdown and immunofluorescence assay.Resultsm6A levels were increased in photoaging and were closely associated with the upregulation of KIAA1429 expression. 1331 differentially m6A methylated genes were identified in the UVR group compared with the control group, of which 1192 (90%) were hypermethylated. Gene ontology analysis showed that genes with m6A hypermethylation and mRNA downregulation were mainly involved in extracellular matrix metabolism and collagen metabolism-related processes. Furthermore, KIAA1429 knockdown abolished the downregulation of TGF-bRII and upregulation of MMP1 in UVR-irradiated human dermal fibroblasts (HDFs). Mechanically, we identified MFAP4 as a target of KIAA1429-mediated m6A modification and KIAA1429 might suppress collagen synthesis through an m6A-MFAP4-mediated process.ConclusionsThe increased expression of KIAA1429 hinders collagen synthesis during UVR-induced photoaging, suggesting that KIAA1429 represents a potential candidate for targeted therapy to mitigate UVR-driven photoaging.