Specific Assay Research Articles

Oridonin alleviates inflammation and endoplasmic reticulum stress in pediatric pneumonia via regulating the SIRT1-mediated Wnt/β-catenin signaling pathway.

Pediatric pneumonia is a prevalent and significant health concern worldwide, with elevated morbidity and mortality rates among affected children. This study was designed to elucidate the therapeutic impact of Oridonin (Ori) on pediatric pneumonia and unravel the underlying mechanisms involved. A pediatric infantile pneumonia model was established in mice through intratracheal administration of LPS. Additionally, a cell damage model was created in WI-38 cells by administering LPS. Protein levels were assessed via western blotting, and cell viability was measured with CCK-8. Inflammatory cytokines were quantified through ELISA, and specific assays were employed to evaluate oxidative stress markers. Flow cytometry was utilized to assess cell apoptosis. Ori alleviated lung inflammation, oxidative stress, apoptosis, and endoplasmic reticulum stress (ERS) in LPS-induced pneumonia mice. In addition, Ori increased the viability of LPS-induced pneumonia cells but decreased cell apoptosis. Furthermore, Ori reduced oxidative stress, inflammation, and ERS in LPS-induced pneumonia cells by enhancing SIRT1 to activate the Wnt/β-catenin pathway. This study suggested that Ori inhibited pediatric pneumonia by dampening the inflammatory response, oxidative stress, cell apoptosis, and ERS via the SIRT1/Wnt/β-catenin pathway.

Mutant HTT protein decreases with CAG repeat expansion: implications for therapeutics and bioassays

Abstract Huntington’s disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in the HTT protein. The mutant CAG repeat is unstable and expands in specific brain cells and peripheral tissues throughout life. Genes involved in the DNA mismatch repair pathways, known to act on expansion, have been identified as genetics modifiers, therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of disease progression. In the context of an expanded CAG repeat, the HTT pre-mRNA can be alternatively processed to generate the HTT1a transcript, that encodes the aggregation prone and highly pathogenic HTT1a protein. This may be a mechanism through which somatic CAG repeat expansion exerts its pathogenic effects, as the longer the CAG repeat, the more HTT1a and HTT1a is produced. The allelic series of knock-in mouse models: HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175 with polyglutamine expansions of 20, 50, 80, 111 140 and ∼190 can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. By western blot of cortical lysates, we found that mutant HTT levels decreased with increasing CAG repeat length; mutant HTT was only 23% and 10% of wild-type levels in CAG140 and zQ175 cortices, respectively. To identify the optimal bioassays for detecting the full-length HTT and HTT1a isoforms, we interrogated the pairwise combinations of seven well-characterized antibodies on both the ‘homogeneous time resolved fluorescence’ and ‘Meso Scale Discovery’ platforms. We tested 32 assays on each platform to detect ‘full-length mutant HTT’, HTT1a, ‘total mutant HTT’ (full-length HTT and HTT1a) and ‘total full-length HTT’ (mutant and wild type). None of these assays recapitulated the full-length mutant HTT levels as measured by western blot. We recommend using isoform- and species- specific assays that detect either full-length mutant HTT, HTT1a or wild-type HTT as opposed to those that detect more than one isoform simultaneously. Our finding that as the CAG repeat expands, full-length mutant HTT levels decrease, whilst HTT1a and HTT1a levels increase has implications for therapeutic strategies. If mutant HTT levels in cells containing (CAG)200 are only 10% of wild-type, HTT-lowering strategies targeting full-length HTT at sequences 3’ to intron 1 HTT will predominantly lower wild-type HTT, as mutant HTT levels in these cells are already depleted. These data support a therapeutic strategy that lowers HTT1a and depletes levels of the HTT1a protein.

Sandwich immunoassay for adrenomedullin precursor and its practical application.

Adrenomedullin (ADM) is a multifaceted peptide hormone involved in numerous physiological processes, including vascular stability, vasodilation, angiogenesis, and anti-inflammatory responses. The processing of ADM results in several fragments, including midregional proadrenomedullin (MR-proADM), and glycine-extended ADM (ADM-Gly) and bioactive ADM (bio-ADM). MR-proADM, the stable ADM fragment, and bio-ADM, the active form of ADM with a short half-life, have been shown to be potent biomarkers in a variety of pathologies. ADM-Gly, the direct precursor of bio-ADM, is a predominant form in human plasma, but remains less understood and least investigated. This study presents the development of a specific immunoluminometric assay for the quantification of ADM-Gly and offers a robust one-step approach for large-scale sample screening. Applied to human and rodent plasma, it elucidates the release kinetics and plasma half-life of ADM-Gly. Our findings confirm the predominance of ADM-Gly in healthy individuals and its significant release under pathological conditions. Our immunoluminometric assay enables precise measurement of ADM-Gly, advancing research into ADM-related pathophysiology and supporting its use as a biomarker and therapeutic target in various diseases.

Abstract A013: Quality control of the biobanked matched tissue: Blood samples for the development of diverse liquid based molecular assays

Abstract The availability of high-quality matched biological samples (tumor-blood) is the major limiting factor for research and development of novel, liquid biopsy-based assays in oncology. Frequently, ongoing specifically timed collections of blood samples are required. De-identified tissue blocks (formalin-fixed, paraffin embedded (FFPE) or flash frozen) matched with patient's blood samples collected in fixative-containing (Streck) of EDTA containing tubes, were collected at worldwide geographic locations and transferred to biobanking facility (Reference Medicine, Phoenix, AZ). Additional blood samples were collected at various times regarding the cancer surgery. Plasma and buffy coat samples were locally prepared and shipped to the biobank. A subset of blood samples without matched tissues from healthy donors were also collected. All samples were shipped under strict transport conditions. Quality control performed at the biobanking facility included pathology review and nucleic acids quality assessment. A total of over 3,000 cases were collected and provided to end-users working on various assays based on cfDNA (e.g. monitoring for minimal residual disease or multi-cancer early detection). Immuno-reactivity to various antigens in samples collected in Streck and EDTA tubes (from 50 patients) was assessed by peptide microarray binding assay. Feedback information on the quality of samples was shared in 900 cases. Approximately 4% of cases had failed internal pathology review, due to poor preservation, lack of adequate tissue composition, or low tumor volume and 11% failed internal nucleic acid quality assessment (DNA concetration and integrity). The blood collection tubes had no significant impact on serologic testing using peptide microarary assay. Customer feedback data showed that overall success rate exceeded 85%. Type of blood collection tubes (Streck vs EDTA) had no impact on serologic testing and studies looking at predicted neoantigens based on NGS data can be confirmed using peptide epitope arrays. Blood collected for NGS works without compromise on peptide arrays. Future validation of other collection tubes for blood or other types of fluids is needed to confirm their adequacy for specific assays. Citation Format: Zoran Gatalica, Phillip Stafford, Chris Diehnelt, Inga Rose. Quality control of the biobanked matched tissue: Blood samples for the development of diverse liquid based molecular assays [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A013.

Synthesis and Anti-Inflammatory and Analgesic Potentials of Ethyl 2-(2,5-Dioxo-1-Phenylpyrrolidin-3-yl)-2-Methylpropanoate

Background/Objectives: Inflammation and analgesia are two prominent symptoms and often lead to chronic medical conditions. To control inflammation and analgesia, many marketed drugs are in practice but the majority of them have severe side effects. Methods: This study involved the synthesis of a pivalate-based Michael product and evaluated it for in vitro COX-1, COX-2, and 5-LOX inhibitory potentials using specific assays. Molecular docking studies were also assessed. Based on the in vitro results, the compound was also subjected to in vivo anti-inflammatory and antinociceptive studies. Results: The pivalate-based Michael product (MAK01) was synthesized by an organocatalytic asymmetric Michael addition of ethyl isobutyrate to N-phenylmaleimide with an isolated yield of 96%. The structure of the compound was confirmed through 1H and 13C NMR analyses. The observed IC50 values for COX-1, COX-2, and 5-LOX were 314, 130, and 105 μg/mL, respectively. The molecular docking studies on the synthesized compound showed binding interactions with the minimized pockets of the respective enzymes. In a carrageenan model, a percent reduction in edema when administered at 10 mg/kg (a reduction of 33.3 ± 0.77% at the second hour), 20 mg/kg (a reduction of 34.7 ± 0.74% at the second hour), and 30 mg/kg (a reduction of 40.58% ± 0.84% after the fifth hour) was observed. The compound showed a significant response at concentrations of 50, 100, and 150 mg/kg with latency times of 10.32 ± 0.82, 12.16 ± 0.51, and 12.93 ± 0.45 s, respectively. Conclusion: In this study, we synthesized a pivalate-based Michael product for the first time. Moreover, based on its rationality and potency, it was found to be an effective future medicine for the management of analgesia and inflammation.

An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products

The expression of therapeutic genes is critical for the efficacy of gene therapy products. However, existing methods such as immunological analysis at the protein level or reverse-transcription PCR at the RNA level are unable to accurately quantify the expression activity of the target gene. Herein, an in vitro RNA editing-based reporter assay was developed to detect specific mRNA. The designed sensor RNA could specifically identify the target mRNA, and the reporter gene was activated in a dose-dependent manner because of RNA editing mediated by endogenous adenosine deaminases acting on RNA. Of note, all sensors that targeted different regions, including the gene of interest, tag sequence, and 3′ untranslated region, showed a dose-dependent response pattern. The sensor reporter assay, which was used for quantifying the transcriptional activity of recombinant adeno-associated virus-based gene therapy products, revealed excellent performance in terms of assay specificity, precision (inter-assay relative standard deviation < 15%), accuracy (90–115% recovery), and linearity (R2 > 0.99). The reporter assay could also be employed for other gene therapy vectors, including mRNA and recombinant lentivirus. Thus, a robust and reliable platform was developed for assessing the transcriptional activity of therapeutic genes, thereby offering a powerful tool for the quality control of gene therapy products.

Mechanisms of fibrinogen trans-activation of the EGFR/Ca2+ signaling axis to regulate mitochondrial transport and energy transfer and inhibit axonal regeneration following cerebral ischemia.

Ischemic stroke results in inhibition of axonal regeneration but the roles of fibrinogen (Fg) in neuronal signaling and energy crises in experimental stroke are under-investigated. We explored the mechanism of Fg modulation of axonal regeneration and neuronal energy crisis after cerebral ischemia using a permanent middle cerebral artery occlusion (MCAO) rat model and primary cortical neurons under low glucose-low oxygen. Behavioral tests assessed neurological deficits; immunofluorescence, immunohistochemistry, and Western-blot analyzed Fg and protein levels. Fluo-3/AM fluorescence measured free Ca2+ and ATP levels were gauged via specific assays and F560nm/F510nm ratio calculations. Mito-Tracker Green labeled mitochondria and immunoprecipitation studied protein interactions. Our comprehensive study revealed that Fg inhibited axonal regeneration post-MCAO as indicated by reduced GAP43 expression along with elevated free Ca2+, both suggesting an energy crisis. Fg impeded mitochondrial function and mediated impairment through the EGFR/Ca2+ axis by trans-activating EGFR via integrin αvβ3 interaction. These results indicate that the binding of Fg with integrin αvβ3 leads to the trans-activation of the EGFR/Ca2+ signaling axis thereby disrupting mitochondrial energy transport and axonal regeneration and exacerbating the detrimental effects of ischemic neuronal injury.

Highly specific multiplex DNA methylation detection for liquid biopsy of colorectal cancer

BackgroundCirculating tumor DNA (ctDNA) has emerged as a useful biomarker for cancer detection and prognosis. In this study, we developed a strategy for developing a highly specific multiplex qPCR assay to detect methylated ctDNA in the blood of colorectal cancer (CRC) patients and investigated the potential use for the detection and prognosis of CRC. MethodsBisulfite conversion and amplicon sequencing were used to confirm potential CRC-specific DNA methylation markers. The selected DNA methylation candidates were validated by qMSP. The six best-performing markers were used to develop a new single-tube multiplex quantitative methylation-specific PCR assay (mqMSP). The mqMSP assay was applied to analyze plasma samples from 114 CRC patients, 47 patients with advanced adenoma, 45 patients with benign polyps, and 57 healthy controls. The clinical performance of the assay and associations with clinical outcomes were assessed. ResultsSix DNA methylation biomarkers were confirmed to be specifically hypermethylated in CRC tumor tissues. The newly developed mqMSP assay detected CRC with extremely high specificity (specificity of 98.2 %, with sensitivity of 67.5 %). The detection rate of ctDNA was significantly correlated with tumor size and clinical stage, with ctDNA methylation levels in the blood markedly increased with larger tumor size, poor differentiation, and advanced stage. Moreover, high preoperative methylated ctDNA level was associated with worse recurrence-free survival and overall survival. ConclusionWe provided a strategy for identification of multiple highly-specific DNA methylation markers for designing multiplex DNA methylation assays for liquid biopsies of CRC. The newly developed assay has potential for CRC early detection, and prognosis.

Development of colorimetric one-step reverse transcription loop-mediated isothermal amplification for rapid and visualized detection of tomato necrotic ringspot virus

Tomato necrotic ringspot virus (TNRV) stands out as one of the most destructive viruses affecting chili and tomato crops in Thailand. TNRV causes typical necrotic and chlorotic ringspot symptoms on both leaves and fruits. This study focused on the development of a colorimetric one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and visualized virus detection in different host plants, including chili, tomato, and physalis, a weed found near tomato fields. The new set of LAMP primers was designed based on the sequence of the non-structural (NSs) protein and nucleocapsid (N) protein genes of TNRV. The assay was optimized to identify the optimum isothermal temperature and incubation period. Positive reactions were identified by a color change from pink to yellow, while negative reactions remained pink where the assay worked well at 65 °C for 30 min. Specificity and sensitivity assays were conducted. The results revealed that the optimal incubation time for detecting LAMP products, along with colorimetric changes, was 30 min at 65 °C. The specificity assay demonstrated where LAMP primers did not cross-react with other orthotospoviruses like capsicum chlorosis virus (CaCV), melon yellow spot virus (MYSV) and watermelon silver mottle virus (WSMoV). The sensitivity assay indicated that RT-LAMP detected TNRV from RNA diluted up to 10−7 (1.0 fg/μL). Evaluation of the colorimetric one-step RT-LAMP assay on naturally infected tomato, chili, and physalis samples confirmed its successful detection of TNRV.

Challenges and prospects of cell-free DNA in precision oncology

Cell-free DNA (cfDNA), predominantly derived from apoptotic or necrotic cells, is emerging as a potential biomarker due to non-invasiveness and cost-effectiveness1. Additionally, another type of cfDNA such as cell-free mitochondrial DNA (cf-mtDNA) that is released from cells under stress or damage condition, may play a key role in the immune system. Thus, the presence of cfDNA in the circulation of cancer patients with differential observations between the cancer and the healthy individuals may be used to improve diagnosis by liquid biopsies2. In the past decades, liquid biopsy has been significantly advanced, particularly in detecting cfDNA, which has opened a new research era in cancer diagnostics worldwide. However, there is a big hindrance in integrating cfDNA analysis with clinical diagnosis, For example, a high rate of false positives is caused by insensitive assays, which may be improved by refining the processing of sequencing data. That is due to the technical differences of feature analysis (e.g., fragmentomics and methylation patterns) and their heterogeneity limited the application of cfDNA in the clinics3–5. Currently, there are many efforts on the way to solve the problems. Especially, the top priority is to enhance assay sensitivity and specificity and to adapt analytical techniques in oncology.Nowadays, liquid biopsy technology based on circulating free DNA (cfDNA) brings new hope for future clinical applications such as minimally invasive early screening for tumors, with the key to effectiveness lying in the integration of next-generation sequencing (NGS) with advanced separation technologies. A comparison between the RNA expression datasets and the cfDNA fragment frequencies has revealed distinct genomic characteristics between cancer patients and healthy individuals. Research has found that there are specific fragmentation patterns of cfDNA in the promoter regions of some highly expressed genes6,7. Notably, cfSort is the first supervised tissue deconvolution approach powered by deep learning, leveraging a high-resolution methylation atlas from 521 non-cancer tissue samples across 29 major human tissue types. This model accurately identifies the origin of cfDNA, demonstrating the clinical value of sequencing data in disease diagnosis and treatment monitoring8. In this perspective, we outline the current trend of cfDNA research and highlight its potential limitations in oncology (Fig. 1). It provides valuable insights for cancer diagnosis and management which may help pave the way for the future adoption of cfDNA in oncology.

Capsular Polysaccharide Production in Bacteria of the Mycoplasma Genus: A Huge Diversity of Pathways and Synthases for So-Called Minimal Bacteria.

Mycoplasmas are wall-less bacteria with many species spread across various animal hosts in which they can be pathogenic. Despite their reduced anabolic capacity, some mycoplasmas are known to secrete hetero- and homopolysaccharides, which play a role in host colonization through biofilm formation or immune evasion, for instance. This study explores how widespread the phenomenon of capsular homopolysaccharide secretion is within mycoplasmas, and investigates the diversity of both the molecules produced and the synthase-type glycosyltransferases responsible for their production. Fourteen strains representing 14 (sub)species from four types of hosts were tested invitro for their polysaccharide secretion using both specific (immunodetection) and nonspecific (sugar dosage) assays. We evidenced a new, atypical homopolymer of β-(1 → 6)-glucofuranose (named glucofuranan) in the human pathogen Mycoplasma (M.) fermentans, as well as a β-(1 → 6)-glucopyranose polymer for the turkey pathogen M. iowae and galactan (β-(1 → 6)-galactofuranose) and β-(1 → 2)-glucopyranose for M. bovigenitalium infecting ruminants. Sequence and phylogenetic analyses revealed a huge diversity of synthases from varied Mycoplasma species. The clustering of these membrane-embedded glycosyltransferases into three main groups was only partially correlated to the structure of the produced homopolysaccharides.

Development of novel digital PCR assays for the rapid quantification of Gram-negative bacteria biomarkers using RUCS algorithm

Rapid and accurate identification of bacterial pathogens is crucial for effective treatment and infection control, particularly in hospital settings. Conventional methods like culture techniques and MALDI-TOF mass spectrometry are often time-consuming and less sensitive. This study addresses the need for faster and more precise diagnostic methods by developing novel digital PCR (dPCR) assays for the rapid quantification of biomarkers from three Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.Utilizing publicly available genomes and the rapid identification of PCR primers for unique core sequences or RUCS algorithm, we designed highly specific dPCR assays. These assays were validated using synthetic DNA, bacterial genomic DNA, and DNA extracted from clinical samples. The developed dPCR methods demonstrated wide linearity, a low limit of detection (∼30 copies per reaction), and robust analytical performance with measurement uncertainty below 25 %. The assays showed high repeatability and intermediate precision, with no cross-reactivity observed. Comparison with MALDI-TOF mass spectrometry revealed substantial concordance, highlighting the methods’ suitability for clinical diagnostics.This study underscores the potential of dPCR for rapid and precise quantification of Gram-negative bacterial biomarkers. The developed methods offer significant improvements over existing techniques, providing faster, more accurate, and SI-traceable measurements. These advancements could enhance clinical diagnostics and infection control practices.

Assessment of the humoral immunity against diphtheria, tetanus, and hepatitis B among children with acute lymphocytic leukemia

Background: Approximately all systemic therapies for childhood affect the immune system. The behavior of the immune system in leukemia patients following chemotherapy is not yet clearly defined. The probability of vaccination failure and the need for revaccination remain challenging for these patients. Aim: To evaluate the humoral immunity against diphtheria, tetanus, and hepatitis B in children with acute lymphocytic leukemia (ALL) immediately and 6 months after chemotherapy. Materials and Methods: In the present prospective cohort study, 21 patients with ALL referred to Mofid Children’s Hospital were studied immediately and 6 months after chemotherapy. Serum samples were collected from patients, and the levels of immunoglobulins (IgG, IgM, IgE, and IgA) antibodies against diphtheria, tetanus, and hepatitis B were determined using specific enzymelinked immunosorbent assay kits. The obtained data were analyzed using Statistical Package for Social Sciences 21 software. Results: A total of 13 males and 8 females with an average age of 8.6 ± 2.5 years were included in the present study. Six months after chemotherapy, the mean level of IgG, IgM, IgE, and IgA displayed an increase of 563.1 units in IgG, 11 units in IgM, 11.3 units in IgE, and 5 units in IgA levels. Moreover, data revealed that 6 months after chemotherapy, the mean level of IgG antibodies displayed an increase of 7.09, 3.43, and 1.03 units against hepatitis B, diphtheria, and tetanus, respectively. A significant relationship was found between the antibody level against diphtheria and the age group of the patients (p = 0.003). Conclusion: Humoral immune status was boosted after 6 months of chemotherapy, though all patients had some extent of lasting immune dysfunction. We indicate that survivors of childhood cancer have ongoing humoral immunological defects and may remain at risk for infectious complications after completion of therapy. Relevance for Patients: The present study indicated that systemic therapies for pediatrics with leukemia affect the immune system. Pediatrics with leukemia may remain at risk for infectious complications after completion of therapy.

Wastewater-Based Epidemiology of Influenza Viruses: a systematic review

Abstract Background Influenza viruses (IVs) are responsible for annual epidemic outbreaks during the winter months. Beyond conventional surveillance systems, the circulation of these pathogens can be monitored using Wastewater-Based Epidemiology (WBE), an epidemiological tool that describes the health status of an entire community by analyzing the presence of pathogens within wastewater. Systematically reviewing existing experiences in this emerging field is crucial to highlight the best practices to establish a WBE surveillance system providing useful information for public health actions. Methods The study protocol for this systematic review has been registered on the PROSPERO website (registration ID: CRD42024532435). Three academic databases (PubMed, Web of Science and Scopus) were accessed to collect studies examining the presence of IVs in wastewater. Studies included were those reporting quantitative measures of viral concentration or detailing molecular/phylogenetic characterization of IVs in wastewater. Results Databases were searched on July 8th, 2024. After de-duplication and screening of the 400 remaining records, 42 were included in the review. IVs detection in wastewater was reported in all papers except from one, although with high variability in terms of positivity titers and concentration levels. More than half of the collected papers (52.4%) evaluated association between environmental viral concentration and clinical data, generally showing concordance between the two aspects. Moreover, 28.6% of papers described subtyping of IVs in wastewater through different methods (specific PCR assays being the most used). Conclusions WBE is a promising approach for surveilling viral circulation, and IVs represent a high value target for implementing this innovative system. Systematically reviewing previous experiences can be useful to obtain information for the establishment of a novel environmental surveillance system of Influenza Viruses Key messages • Nucleic acids belonging to Influenza Virus are detectable in wastewater, even though we report high levels of variability in sampling techniques, laboratory methods and data management systems. • Association between environmental data and number of clinical cases is reported to be generally strong in collected papers; subtyping of IVs present in wastewater is also feasible.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

Development of a new tool to detect Melampsora medusae f.sp. tremuloidae causing rust disease on Populus tremuloides.

Melampsora medusae f. sp. tremuloidae is a quarantine organism for the EU. In North America, this fungus causes rust disease on Populus tremuloides. In Europe, Populus tremula, an aspen closely related to P. tremuloides, is widespread and plays an important ecological role. Introduction of M. medusae f. sp. tremuloidae into Europe could be a major risk if this forma specialis could evolve and become virulent on P. tremula. To date no PCR-based assay exists to specifically detect M. medusae f. sp. tremuloidae. In this study, a sensitive and specific real-time PCR assay has been developed based on the 28S rDNA. The assay proved to be reliable using many real-time PCR kits and platforms. It can be used to monitor the introduction and the spread of M. medusae f. sp. tremuloidae in the context of phytosanitary regulations.

Evaluation of Multiplex loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis complex from clinically suspected cases of pulmonary tuberculosis

Tuberculosis (TB) is the second leading cause of death from a single infectious agent worldwide. Bangladesh ranks 7th among the 30 high TB burdened countries in the world. Accurate detection of Mycobacterium tuberculosis complex (MTBC) is challenging for developing countries as most of the resource poor settings are not suitable to perform molecular techniques.The purpose of the study was to compare the multiplex TB-LAMP assay with MTB/NTM qPCR, culture, Z-N staining, and fluorescence microscopy in order to assess the effectiveness of the LAMP assay for detecting cases of pulmonary tuberculosis.This research work was done from March 2022 to February 2023. Fulfilling the inclusion criteria 130 sputum samples were collected. TB-LAMP assay, qPCR, culture in L-J media, Z-N staining and fluorescence microscopy were performed.Out of 130 samples qPCR detected MTBC in 56.92 % cases and TB-LAMP detected 53.85 %. MTBC was detected by culture 46.15 %, by Fluorescence microscopy 40.77 % and Z-N staining 36.92 %. TB-LAMP detected 16.93 % more cases than Z-N staining and 13.08 % more cases than fluorescence microscopy. The sensitivity, specificity, positive, and negative predictive values of multiplex-LAMP assay were 95 %, 81.4 %, 81.4 % and 95 % respectively considering culture as a gold-standard. MTBC negative culture samples (18.57 %) showed positivity by LAMP assay as well as by qPCR. This study detected 7.69 % non-tuberculous mycobacteria (NTM) by qPCR. All NTM positive samples were negative by TB-LAMP.TB-LAMP is an easy to perform, cost-effective, reliable assay with high sensitivity and specificity. World Health Organization recommended TB-LAMP as a rapid molecular test for rapid detection of tuberculosis and as replacement of microscopy in resource poor settings/hard to reach areas. Bangladesh being a high TB burden country it is essential to implement TB-LAMP to achieve End TB Strategy by 2035.

Phytochemical Characterization and Assessment of the Wound Healing Properties of Three Eurasian Propolis

Objectives: The objective of this study is to evaluate the wound healing potential of Eurasian propolis by analyzing the phytochemical profile and the biological effects of three representative propolis samples. Methods: Specific colorimetric assays were used to estimate the total phenolic and flavonoid contents and the triterpenoids content. Some of the main components of Eurasian propolis (pinocembrin, pinobanksin, CAPE, chrysin and galangin) were analyzed using HPLC-DAD. Scavenging activity and total antioxidant capacity were assessed through DPPH and ORAC assays, respectively. Human keratinocyte, fibroblast, and monocytic cell lines were used for the biological in vitro analyses. The direct wound healing properties were tested through scratching assays and ELISA kits for the assessment of the production of growth factors (FGF-7, Latency Associated Peptide-LAP), while the indirect effects were evaluated through the estimation of the levels of MMP9, IL-1β, IL-8, and TNF-α using ELISA kits together with a cell-free test on the inhibition capacity on collagenases. Network Pharmacology analysis was employed to further explore possible mechanisms of the action of propolis on the healing process. Results: The analyses confirmed the high phenolic content of Eurasian propolis (142.50–211.30 mg GAE/g), dominated by flavonoids (95.50–196.80 mg Galangin Equivalents/g), and terpenes (431.50–650.00 mg β-sitosterol Equivalents/g), while also verifying the significant antioxidant (4.9–8.9 mM/g Trolox Equivalents) and antiradical (DPPH IC50 26.1–54.4 μg/mL) activities. The samples showed indirect wound healing properties by mitigating inflammation and remodeling (reduced IL-1β and MMP9) and potentially modulating the immune response (upregulated IL-8). In vitro studies confirmed these effects, demonstrating decreased MMP9 production and collagenase inhibition when cells were co-treated with propolis and a stressor. Propolis also suppressed IL-1β release in fibroblasts, although its impact on TNF-α was inconclusive. Notably, co-treatment upregulated IL-8 in monocytes, suggesting a potential immunomodulatory role. Conclusions: Eurasian propolis may not directly stimulate cell proliferation during wound healing. Its anti-inflammatory and immunomodulatory properties could indicate an indirect contribution in helping the process.

Optimisation of induction and purification protocols of recombinant 22.6kDa tegumental protein of Schistosoma spindale inprokaryotic vector

Schistosomosis is a prevalent zoonotic parasitic disease affecting both humans and animals on a global scale, with an estimated 165 million cattle and 200 million people impacted worldwide. Eventhough, serological methodologies designed for the identification of specific antibodies targeting parasitic antigens are esteemed for their high sensitivity, there are criticisms due to their incapacity to reliably indicate active infection, inability to correlate with the intensity of infection and lack of specificity. Enhancing the specificity of serological assays presents a significant challenge, primarily attributable to the identification and synthesis of specific antigens. In addressing these limitations, recombinant technology with specific immunogenic proteins as candidate antigens emerges as a viable alternative. In this study, induction of 22.6 kDa recombinant tegument protein of Schistosoma spindale was achieved using 0.6 mM concentration of IPTG at 37°C for four hours. Nickel chelating affinity chromatography was employed for protein purification, yielding maximum protein concentration at 75mM elution. Subsequently, the dialysis technique was employed to remove contaminants, while lyophilisation method was employed for protein concentration. The protein concentration post dialysis was measured at 0.220 mg/mL, while lyophilisation resulted in a concentration of 2mg/m Keywords: 22.6 kDa tegumental protein, Schistosoma spindale, dialysis, lyophilisation