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Environmental DNA (eDNA) once shed can exist in numerous states with varying behaviors including degradation rates and transport potential. In this study, we consider three states of eDNA: (1) a membrane-bound state referring to DNA enveloped in a cellular or organellar membrane, (2) a dissolved state defined as the extracellular DNA molecule in the environment without any interaction with other particles, and (3) an adsorbed state defined as extracellular DNA adsorbed to a particle surface in the environment. Capturing, isolating, and analyzing a target state of eDNA provides utility for better interpretation of eDNA degradation rates and transport potential. While methods for separating different states of DNA have been developed, they remain poorly evaluated due to the lack of state-controlled experimentation. We evaluated the methods for separating states of eDNA from a single sample by spiking DNA from three different species to represent the three states of eDNA as state-specific controls. We used chicken DNA to represent the dissolved state, cultured mouse cells for the membrane-bound state, and salmon DNA adsorbed to clay particles as the adsorbed state. We performed the separation in three water matrices, two environmental and one synthetic, spiked with the three eDNA states. The membrane-bound state was the only state that was isolated with minimal contamination from nontarget states. The membrane-bound state also had the highest recovery (54.11 ± 19.24%), followed by the adsorbed state (5.08 ± 2.28%), and the dissolved state had the lowest total recovery (2.21 ± 2.36%). This study highlights the potential to sort the states of eDNA from a single sample and independently analyze them for more informed biodiversity assessments. However, further method development is needed to improve recovery and reduce cross-contamination., Environmental DNA, 5 (3), ISSN:2637-4943

In 2020 and 2021 we detected in Catalonia (NE Spain) six new episodes of mortality or presence of dying specimens in introduced Mediterranean painted frogs (Discoglossus pictus). All affected individuals were positive to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). Histological examinations showed intense involvement of the superficial layers of the epidermis and qPCR analyses yielded infection loads greater than 100 000 genomic equivalents of zoospores. These results suggest that this introduced species could behave as a Bd ‘supershedder’, and recommend to control its expansion due to its important role in the dynamic of chytridiomycosis.

RESUMEN El objetivo de este estudio fue determinar la presencia del Virus Papiloma Humano (VPH) tipo 16 y 18 en biopsias de tejido mamario parafinado de pacientes con diagnóstico clínico de cáncer de mama. Se analizaron 32 biopsias de cáncer de mama embebidas en parafina para detectar el ADN de VPH mediante PCR en tiempo real, los iniciadores estuvieron dirigidos al gen E6. Se evaluaron el tipo histológico, grado histológico y la sobreexpresión de C-erB2 y Ki-67 mediante inmunohistoquímica. El 84,38% (27) fueron positivos para VPH, el 25% (8) fueron positivos para VPH-16 y el 59,38% (19) para VPH-18. El 15,63% (5) de las muestras presentaron infección mixta. Se evidenció la sobrexpresión de C-erbB2 y Ki-67 en 6,25% (2) de las muestras positivas para VPH-16 y 15,63% (5) de las muestras positivas para VPH-18. Se detectó ADN de VPH-16 y VPH-18 en las muestras de biopsias analizadas mediante PCR en tiempo real. ABSTRACT The aim of this study was to determine the presence of Human Papillomavirus (HPV) type 16 and 18 in biopsies of paraffin-embedded breast tissue from patients with clinically diagnosed breast cancer. 32 paraffin-embedded breast cancer biopsies were analyzed in order to detect HPV DNA by real-time PCR, the primers were directed at the E6 gene. The histological type, histological grade and overexpression of C-erB2 and Ki-67 were evaluated by immunohistochemistry. 84.38% (27) of the samples were positive for HPV, 25% (8) were positive for HPV-16 and 59.38% (19) were positive for HPV-18. Mixed infection was found in 15.63% (5) of the samples. Overexpression of C-erbB2 and Ki-67 was seen in 6.25% (2) of the samples positive for HPV-16 and in 15.63% (5) samples positive for HPV-18. HPV-16 and HPV-18 DNA was detected in the biopsy samples analyzed by real-time PCR.

Mitochondrial DNA (mtDNA) damage is closely associated with typical diseases of aging, such as Alzheimer’s or Parkinson’s disease, and other health conditions, such as infertility. This damage manifests in reduced mitochondrial copy number and deletion mutations in mtDNA. Consequently, the analysis of mitochondrial damage by determining the parameters copy number and deletion ratio using quantitative real-time PCR (qPCR) is of interest for clinical diagnostics. To bring the findings from research into laboratory practice, a suitable and reliable process is needed, which must be thoroughly validated. This process includes the software used for the analysis, which must meet extensive regulatory and process requirements. Existing software does not adequately implement the requirements of laboratories and, in particular, does not provide direct support for the calculation of the aforementioned mtDNA parameters. The paper discusses the development of a new software-based analysis workflow that is designed specifically for laboratories to help with the calculation of mtDNA parameters. The software was developed using the User-Centered Design method and is based on the recently introduced prototype, “PlateFlow”. Initial user tests provide positive feedback. In the future, this workflow could form the basis for validations of mitochondrial tests in medical laboratories.

Quantitative PCR (qPCR) has become a widely used technique for bacterial quantification. The affordability, ease of experimental design, reproducibility, and robustness of qPCR experiments contribute to its success. The establishment of guidelines for minimum information for publication of qPCR experiments, now more than 10 years ago, aimed to mitigate the publication of contradictory data. Unfortunately, there are still a significant number of recent research articles that do not consider the main pitfalls of qPCR for quantification of biological samples, which undoubtedly leads to biased experimental conclusions. qPCR experiments have two main issues that need to be properly tackled: those related to the extraction and purification of genomic DNA and those related to the thermal amplification process. This mini-review provides an updated literature survey that critically analyzes the following key aspects of bacterial quantification by qPCR: (i) the normalization of qPCR results by using exogenous controls, (ii) the construction of adequate calibration curves, and (iii) the determination of qPCR reaction efficiency. It is primarily focused on original papers published last year, where qPCR was applied to quantify bacterial species in different types of biological samples, including multi-species biofilms, human fluids, and water and soil samples., Christina A. Muzny, MD, MSPH, Christopher M. Taylor, PhD, Nuno Cerca, PhD and Ângela Lima, BSc, are currently funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation., info:eu-repo/semantics/publishedVersion

11 Pág. Departamento de Reproducción Animal​, Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 μM), and the effect of cortisol addition (0.25 μM, 0.5 μM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1., This research was supported by the projects PID2019-108320RJ-I00, IJCI-2015-24380, PID2020-116531RB-I00 and RYC2020- 028615-I, funded by MCIN/AEI/10.13039/501100011033 (Spain) and FEDER funds (EU). MRC is supported by the Government of Spain, Ministry of Education, Culture and Sports (Training Programme for Academic Staff FPU15/06029). JG was supported by the Govern ment of Catalonia (Generalitat de Catalunya, AGAUR), co-financed with the European Social Found (2018FI_00236).

Aims Antibiotic-resistant bacteria affect human and animal health. Hence, their environmental spread represents a potential hazard for mankind. Livestock farming is suspected to be a key factor for spreading antibiotic resistance; consumers expect organic farming to imply less environmental health risk. This study aimed to assess the role of manure from organic and conventional farms for spreading antimicrobial resistance (AMR) genes. Methods and Results AMR-genes—namely tet(A), tet(B), tet(M), sul2 and qacE/qacEΔ1 (potentially associated with multiresistance) were quantified by qPCR. Antimicrobial use during the study period was qualitatively assessed from official records in a binary mode (yes/no). Median concentrations were between 6.44 log copy-equivalents/g for tet(A) and 7.85 for tet(M) in organic liquid manure, and between 7.48 for tet(A) and 8.3 for sul2 in organic farmyard manure. In conventional manure, median concentrations were 6.67 log copy-equivalents/g for sul2, 6.89 for tet(A), 6.77 for tet(B) and 8.36 for tet(M). Integron-associated qac-genes reached median concentrations of 7.06 log copy-equivalents/g in organic liquid manure, 7.13 in conventional manure and 8.18 in organic farmyard manure. The use of tetracyclines or sulfonamides increased concentrations of tet(A) and tet(M), or of sul2, respectively. Comparing farms that did not apply tetracyclines during the study, the relative abundance of tet(A) and tet(M) was still higher for conventional piggeries than for organic ones. Conclusions Relative abundances of AMR genes were higher in conventional farms, compared to organic ones. Antibiotic use was linked to the relative abundance of AMR-genes. However, due to the bacterial load, absolute concentrations of AMR-genes were comparable between fertilizers of organic and conventional farms. Significance and Impact of Study To our knowledge, this is the first absolute quantification of AMR-genes in manure from organic farms. Our study underlines the importance of long-term reduction in the use of antimicrobial agents in order to minimize antibiotic resistance.

Escherichia coli serogroup O157 is the main causative agent of several intestinal and extra-intestinal foodborne diseases in humans through consumption of low-dose contaminated foods such as milk, beef, and vegetables. To date, studies regarding the quantitative prevalence of E. coli O157 in foods are so limited. Therefore, this study aimed to evaluate the quantitative prevalence rate of E. coli serogroup O157 in raw milk (n = 144), vegetable salad (n = 174), and minced beef samples (n = 108) using the real-time qPCR SYBR green melting curve method targeting the rfbA gene. First, we evaluated the method and found a sensitive and specific qPCR assay with 1 log of CFU/ml detection limit to detect E. coli O157 (Tm = 80.3 ± 0.1°C). About 2.77%, 10.18%, and 9.19% of raw milk, minced beef, and vegetable salad samples, respectively, were contaminated with E. coli O157. Minced beef and vegetable salad samples were significantly more contaminated than raw milk samples. Population average of E. coli O157 in raw milk, minced beef, and vegetable salad samples were 2.22 ± 0.57, 3.30 ± 0.40, and 1.65 ± 0.44 log CFU/ml or gr, respectively. Significantly higher levels of population of E. coli O157 were observed in minced beef samples. Minced beef can be regarded as the main food in the transmission of this foodborne pathogen. Routine quantitative rapid monitoring is strongly suggested to be carried out to prevent foodborne diseases caused by E. coli O157.

During the last years, the measurement of minimal residual disease (MRD) has become more and more accepted in the clinical and prognostic assessment of various malignant diseases. In childhood acute leukemic leukemia (ALL) as well, the determination of MRD burden during and after therapy is a crucial factor for the prognosis of an affected patient and further therapeutic steps. Although various methods can be used to measure MRD, the molecular approach, mainly using quantitative real-time polymerase chain reaction (qRT-PCT), is the current gold standard. Different target sequences, such as T-cell receptor and immunoglobulin rearrangements and specific fusion genes are used to monitor a specific blast subpopulation. Based on preferably two or more patient-specific target sequences, disease progression can be estimated. For qRT-PCR, each follow-up bone marrow aspiration is compared to a standard curve of blasts obtained at diagnosis onset, allowing to calculate the current blast burden in the patients' bone marrow. The sensitivity of qRT-PCR ranges up to the detection of one leukemia-type DNA copy in up to 10,000 (10^-4) and in some cases even 1 in 1,000,000 (10^-6) healthy copies. In this project, nine pediatric ALL patients and 17 different MRD targets have been investigated. By measuring dilution series of leukemic blast DNA in healthy DNA with dilution factors from 10^-1 to 10^-6 with qRT-PCR, reliable and reproducible MRD determination was possible. Eight targets reached a sensitivity of 10^-5, also eight a sensitivity of 10^-4 and two targets even 10^-6. Subsequently, MRD measurements were established with digital droplet PCR (ddPCR) as well. Here, the sample to be measured is split into up to 20,000 tiny partitions, thus enabling precise detection and absolute quantification of a PCR product. Eleven of our targets reached a sensitivity of 10^-5, however, among them are two samples with grey-zone results at the dilution of 10^-5. Five targets reached a sensitivity of 10^-4 and one target 10^-6. Unspecific background amplification occurred in both methods and hampered accurate sample determination in one but not the same target per method. Within this project it was possible to visualize all MRD sequences of our nine exemplary patients well and to achieve the same sensitivities with ddPCR as with qRT-PCR. ddPCR mainly offers the advantage to absolutely quantify blasts present at any later follow-up time point without the need for a dilution curve. Further experience in our laboratory and international guidelines for MRD measurement by ddPCR are next necessary steps towards the clinical application of this method.

This study aimed to compare microscopic counting, culture, and quantitative or real-time PCR (qPCR) to quantify sulfate-reducing bacteria in environmental and engineered sludge samples. Four sets of primers that amplified the dsrA and apsA gene encoding the two key enzymes of the sulfate-reduction pathway were initially tested. qPCR standard curves were constructed using genomic DNA from an SRB suspension and dilutions of an enriched sulfate-reducing sludge. According to specificity and reproducibility, the DSR1F/RH3-dsr-R primer set ensured a good quantification based on dsrA gene amplification; however, it exhibited inconsistencies at low and high levels of SRB concentrations in environmental and sulfate-reducing sludge samples. Ultimately, we conducted a qPCR method normalized to dsrA gene copies, using a synthetic double-stranded DNA fragment as a calibrator. This method fulfilled all validation criteria and proved to be specific, accurate, and precise. The enumeration of metabolically active SRB populations through culture methods differed from dsrA gene copies but showed a plausible positive correlation. Conversely, microscopic counting had limitations due to distinguishing densely clustered organisms, impacting precision. Hence, this study proves that a qPCR-based method optimized with dsrA gene copies as a calibrator is a sensitive molecular tool for the absolute enumeration of SRB populations in engineered and environmental sludge samples.

As pest management options, such as nematicides, become more restrictive, developing rootstocks resistant to the citrus nematode is fundamental for citrus production. This study provides an updated methodology to screen for citrus nematode resistance in rootstock-breeding programs. We developed a novel method to extract female citrus nematodes from roots that is suitable for molecular work and a real-time-PCR-based nematode quantification method for Tylenchulus semipenetrans. These procedures allow scaling up screening to high-throughput workflows, increasing the chances of finding rootstock candidates that combine all the desired traits. Our results contribute to the growing literature supporting quantification of nematodes with molecular methods.

The brown planthopper Nilaparvata lugens (Stål) (BPH) is a typical monophagous sucking rice pest. Over the course of their evolution, BPH and its symbionts have established an interdependent and mutually beneficial relationship, with the symbionts being important to the growth, development, reproduction, and variation in virulence of BPH. Yeast-like symbionts (YLS), harbored in the abdomen fat body cells of BPH, are vital to the growth and reproduction of the host. In recent research, the symbionts in BPH have mainly been detected using blood cell counting, PCR, real-time quantitative PCR, and other methods. These methods are vulnerable to external interference, cumbersome, time consuming and laborious. Droplet digital PCR (ddPCR) does not need a standard curve, can achieve absolute quantification, does not rely on Cq values, and is more useful for analyzing copy number variation, gene mutations, and relative gene expression. A rapid detection method for the YLS of BPH based on ddPCR was established and optimized in this study. The results showed that the method’s limits of detection for the two species of YLS (Ascomycetes symbionts and Pichia guilliermondii) were 1.3 copies/μL and 1.2 copies/μL, respectively. The coefficient of variation of the sample repetition was less than 5%; therefore, the ddPCR method established in this study had good sensitivity, specificity, and repeatability. It can be used to detect the YLS of BPH rapidly and accurately.

Objectives: We aimed to assess the performance of the Novodiag® Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections. Methods: A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia. Results: Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for Blastocystis hominis, Entamoeba histolytica and Cyclospora cayetanensis but was lower for Giardia intestinalis (85.2%) and ≤50% for Cystoisospora belli and Dientamoeba fragilis. In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for G. intestinalis, D. fragilis, Cryptosporidium spp. and E. histolytica. Sensitivity was 100% for Enterocytozoon bieneusi, but none of the five samples containing Encephalitozoon spp. were detected. Conclusions: The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR.

Mezenhimske matične/stromalne celice (MSC) so podskupina heterogenih, nehematopoetskih fibroblastom podobnih celic. Njihova fiziološka vloga je vzdrževanje homeostaze tkiv, predvsem s proliferacijo, diferenciacijo in s sproščanjem rastnih dejavnikov in imunomodulatorjev. Zaradi teh edinstvenih karakteristik so MSC privlačne za področje regenerativne medicine, vnetnih bolezni in zdravljenje raka. Eno izmed potencialnih bolezni za zdravljenje z MSC predstavlja kronična degenerativna bolezen osteoartroza (OA). Najbolj očiten sindrom OA je okvara sklepnega hrustanca, ki je posledica degeneracije zunajceličnega matriksa. Zaradi nezmožnosti lastne regeneracije po poškodbi, ki je lahko vzrok za OA, se za zdravljenje sklepnega hrustanca raziskuje uporaba MSC. Te imajo poleg sposobnosti diferenciacije tudi vlogo pri uravnavanju imunskega odziva. MSC namreč delujejo na imunske celice z različnimi imunomodulatornimi učinki posredovanimi s topnimi dejavniki ali neposredno preko medceličnih stikov. Namen magistrske naloge je ugotoviti razlike v izražanju genov za imunomodulatorne molekule v MSC pridobljenih iz različnih tkiv kolka preiskovancev z degenerativnimi spremembami kolka ter tistimi brez, da bi ugotovili, če imajo katere od teh MSC boljši imunomodulatorni potencial. Vzorci so bili pridobljeni med artroskopskim posegom na kolku, in sicer iz treh tkiv sinovije in stebrička trabekularne kosti stegnenice. Primarne MSC smo namnožili v pogojih in vitro ter iz njih izolirali RNA. Pred analizo vzorcev smo optimizirali metodo kvantitativno verižno reakcijo s polimerazo (qPCR) za merjenje izražanja 8 genov za imunomodulatorne molekule kot so indolamin 2.3-dioksigenaza 1 (IDO1), interlevkin 1 beta (IL1B), ligand za programirano celično smrt (PDL1), adhezijski protein vaskularnih celic 1 (VCAM1), hepatocitni rastni dejavnik (HGF), kompleks tkivne skladnosti pri človeku G (HLA-G), s TNF-α induciran protein 6 (TNFAIP6) in antagonist receptorja za interlevkin 1 (IL1RA). Na podlagi optimizacije smo za nadaljnjo analizo izbrali 6 genov (PDL1, VCAM1, HGF, HLA-G, TNFAIP6 in IL1RA). Rezultate pridobljene s qPCR smo nato normalizirali glede na izbran referenčni gen GAPDH in jih primerjali med bolniki z zgodnjo OA in tistimi brez ter med različnimi tkivi. Preverili smo tri hipoteze, in sicer: 1.) izražanje genov za imunomodulatorne molekule se razlikuje med MSC bolnikov z zgodnjo OA v primerjavi s tistimi brez OA, 2.) izražanje genov za imunomodulatorne molekule se razlikuje med MSC iz različnih tkiv bolnikov z zgodnjo OA in 3.) izražanje genov za imunomodulatorne molekule se razlikuje med MSC iz različnih tkiv bolnikov brez OA. Delno smo potrdili le prvo hipotezo za gen HGF, drugi dve pa zavrgli. Gen HGF se približno 4-krat bolj izraža v MSC bolnikov brez OA v primerjavi s tistimi z zgodnjo OA, kar predstavlja pomembno osnovo za nadaljnje raziskave imunomodulatornih učinkov MSC pri OA. Z našo študijo nismo identificirali tkivno-specifičnih MSC z imunomodulatornim potencialom verjetno zaradi premajhnega števila vzorcev, zato predlagamo dodatne študije na večjem številu preiskovancev. Mesenchymal stem/stromal cells (MSCs) are a subset of heterogeneous, non-haematopoietic fibroblast-like cells. Their physiological role is to maintain tissue homeostasis, mainly through proliferation, differentiation, and the release of growth factors and immunomodulators. These unique characteristics make MSCs attractive for regenerative medicine, inflammatory diseases, and cancer treatment. One of the potential diseases for MSC therapy is osteoarthritis (OA), chronic degenerative disease of the joints. The most obvious syndrome of OA is articular cartilage damage resulting from degeneration of the extracellular matrix. Due to the inability of the articular cartilage to regenerate itself after injury, which may be the cause of OA, the use of MSCs for the treatment of articular cartilage is being investigated. In the addition to their differentiation capacity, MSCs also play a role in regulating immune response. MSCs act on immune cells through various immunomodulatory effects mediated by soluble factors or directly via intercellular contacts. The aim of this thesis is to determine the differences in gene expression of immunomodulatory molecules in MSCs obtained from different hip tissues of subjects with and without degenerative hip lesions, to determine if any of these MSCs have a better immunomodulatory potential. Samples were obtained during hip arthroscopy from three tissues of the synovium and the femoral trabecular bone column. Primary MSCs were amplified under in vitro conditions and RNA was isolated from them. Prior to sample analysis, we optimised the quantitative polymerase chain reaction (qPCR) method to measure the expression of 8 genes for immunomodulatory molecules such as indoleamine 2,3-dioxygenase 1 (IDO1), interleukin 1 beta (IL1B), programmed cell death ligand 1 (PDL1), vascular cell adhesion molecule 1 (VCAM1), hepatocyte growth factor (HGF), human leukocyte antigen G (HLA-G), TNF-α-induced protein 6 (TNFAIP6), and interleukin 1 receptor antagonist (IL1RA). Based on optimisation we selected 6 genes for further analysis (PDL1, VCAM1, HGF, HLA-G, TFNAIP6 and IL1RA). The results were then normalised to the selected GAPDH reference gene and compared between patients with and without early OA and between different tissues. Three hypotheses were tested: 1.) gene expression for immunomodulatory molecules differs between MSCs from patients with early OA compared to those without OA, 2.) gene expression for immunomodulatory molecules differs between MSCs from different tissues from patient with early OA and 3.) gene expression for immunomodulatory molecules differs between MSCs from different tissues from patients without OA. Only the first hypothesis was partially confirmed for the HGF gene, while the other two were rejected. The HGF gene is approximately 4 times more highly expressed in MSCs from patient without OA compared to those with early OA, which provides an important basis for further investigation of the immunomodulatory effects of MSCs in OA. Our study did not identify tissue specific MSCs with immunomodulatory potential, probably due to insufficient sample numbers, and we suggest further studies in a larger number of subjects.

Hepatitis B virus (HBV) DNA diagnosis plays a critical role in the transmission of HBV infections. Accurate protocols in HBV detection will reduce the spread of the virus from blood donors. Therefore, this research compared the HBV detecting potentials of three different HBV protocols in the virus detection. Blood from 200 donors at the University college blood bank Ibadan were sampled and tested for HBV using the ABON™ HBsAg Hepatitis B Surface Antigen Tests (strip test), Monolisa TM HBsAg ULTRA (ELISA test) and Huruian biology HBV detection kit (qPCR test). HBV positive samples were genotyped by sequencing to ascertain genotype identity. Results showed that of the 200 samples tested, both ABON™ HBsAg Hepatitis B Surface Antigen Tests and Monolisa TM HBsAg ULTRA (for ELISA) did not record any positive sample. However, Huruian biology HBV detection kit via qPCR recorded a total of twenty-three positive samples. Of the twenty-three samples, twenty were of genotype E while three were of genotype B. The results from this study showed that that qPCR is more accurate in HBV virus detection than ELISA technique and also HBV genotype E is the most common genotype isolated in Nigeria.

Antibiotic-resistant bacteria and antibiotic resistance gene (ARGs) loads dissipate through sewage treatment plants to receiving aquatic environments, but the mechanisms that mitigate the spread of these ARGs are not well understood due to the complexity of full-scale systems and the difficulty of source tracking in downstream environments. To overcome this problem, we targeted a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR), whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs and receiving aquatic ecosystems. We analyzed a large set of physicochemical measurements, concomitant with the cultivation of total and cefotaxime-resistant Escherichia coli, microbial community analyses, and quantitative PCR (qPCR)/digital droplet PCR (ddPCR) quantification of selected ARGs and mobile genetic elements (MGEs). The MABR removed most of the sewage-derived organic carbon and nitrogen, and simultaneously, E. coli, ARG, and MGE levels dropped by approximately 1.5- and 1.0-log unit mL(-1), respectively. Similar levels of E. coli, ARGs, and MGEs were removed in the reservoir, but interestingly, unlike in the MABR, the relative abundance (normalized to 16S rRNA gene-inferred total bacterial abundance) of these genes also decreased. Microbial community analyses revealed the substantial shifts in bacterial and eukaryotic community composition in the reservoir relative to the MABR. Collectively, our observations lead us to conclude that the removal of ARGs in the MABR is mainly a consequence of treatment-facilitated biomass removal, whereas in the stabilization reservoir, mitigation is linked to natural attenuation associated with ecosystem functioning, which includes abiotic parameters, and the development of native microbiomes that prevent the establishment of wastewater-derived bacteria and associated ARGs.IMPORTANCE Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs. We evaluated ARB and ARG dynamics across the raw-sewage-MABR-effluent trajectory, concomitant with evaluation of microbial community composition and physicochemical parameters, in an attempt to identify mechanisms associated with ARB and ARG dissipation. We found that removal of ARB and ARGs in the MABR was primarily associated with bacterial death or sludge removal, whereas in the reservoir it was attributed to the inability of ARBs and associated ARGs to colonize the reservoir due to a dynamic and persistent microbial community. The study demonstrates the importance of ecosystem functioning in removing microbial contaminants from wastewater. Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs.

Rapid water-quality monitoring methods for beach water and sand can be helpful for public health authorities to develop comprehensive beach monitoring programs. In this study, we evaluated the performance of the Enterococcus qPCR (USEPA 1609.1) method for quality monitoring of beach water and sand-porewater at two Niagara region beaches in Ontario, Canada (Lakeside and Sunset Beaches). While the USEPA 1609.1 method has been validated for beach water, its potential for assessing associated beach sands (which may function as a microbial reservoir) has not been fully explored. All beach water samples (n = 168) passed the qPCR quality control (QC). However, only 20 out of 48 (41.7%) sand-porewater samples passed the qPCR QC, potentially due to interference by soluble inhibitors. The proportion of the sand-porewater samples passing QC improved slightly to 63 out of 120 (52.5%) with a prefiltration step to remove sand and other large particles. The faecal indicator density in the sand-porewater, tested in parallel, did not correlate with the beach water faecal indicator density. Comparing beach water data for the same-day Enterococcus qPCR threshold with the previous-day E. coli culturing-based threshold across all beach days tested, Enterococcus qPCR analyses identified 3 (7%) and 7 (16%) false positive/lost beach days for Lakeside and Sunset Beaches, respectively. Additionally, of the total beach days tested, Enterococcus qPCR analyses identified 2 (5%) and 1 (2%) false negative/health-risk days for Lakeside and Sunset Beaches, respectively. Sand-porewater testing analyses identified days when faecal indicators (in the sand) exceeded beach water quality thresholds. Compared with conventional E. coli culturing, use of the same-day qPCR method would result in fewer beach postings and could identify several additional health-risk days (when the beaches may not be posted). Future studies could include additional prefiltration steps or modifications in the Enterococcus qPCR protocol to improve the method’s applicability for sand quality monitoring.

ABCG2 (ATP-binding cassette superfamily G member 2) is a cell membrane pump encoded by the ABCG2 gene. ABCG2 can protect cells against compounds initiating and/or intensifying neoplasia and is considered a marker of stem cells responsible for cancer growth, drug resistance and recurrence. Expression of the ABCG2 gene or its protein has been shown to be a negative prognostic factor in various malignancies. However, its prognostic significance in colorectal cancer remains unclear. Using publicly available data, ABCG2 was shown to be underexpressed in colon and rectum adenocarcinomas, with lower expression compared to both the adjacent nonmalignant lung tissues and non-tumour lung tissues of healthy individuals. This downregulation could result from the methylation level of some sites of the ABCG2 gene. This was connected with microsatellite instability, weight and age among patients with colon adenocarcinoma, and with tumour localization, population type and age of patients for rectum adenocarcinoma. No association was found between ABCG2 expression level and survival of colorectal cancer patients. In wet analysis of colorectal cancer samples, neither ABCG2 gene expression, analysed by RT-PCR, nor ABCG2 protein level, assessed by immunohistochemistry, was associated with any clinicopathological factors or overall survival. An ABCG2-centered protein–protein interaction network build by STRING showed proteins were found to be involved in leukotriene, organic anion and xenobiotic transport, endodermal cell fate specification, and histone methylation and ubiquitination. Hence, ABCG2 underexpression could be an indicator of the activity of certain signalling pathways or protein interactors essential for colorectal carcinogenesis.

Atopic dermatitis (AD) is an inflammatory skin disease with a microbiome dysbiosis towards a high relative abundance of Staphylococcus aureus. However, information is missing on the actual bacterial load on AD skin, which may affect the cell number driven release of pathogenic factors. Here, we combined the relative abundance results obtained by next-generation sequencing (NGS, 16S V1-V3) with bacterial quantification by targeted qPCR (total bacterial load = 16S, S. aureus = nuc gene). Skin swabs were sampled cross-sectionally (n = 135 AD patients; n = 20 healthy) and longitudinally (n = 6 AD patients; n = 6 healthy). NGS and qPCR yielded highly inter-correlated S. aureus relative abundances and S. aureus cell numbers. Additionally, intra-individual differences between body sides, skin status, and consecutive timepoints were also observed. Interestingly, a significantly higher total bacterial load, in addition to higher S. aureus relative abundance and cell numbers, was observed in AD patients in both lesional and non-lesional skin, as compared to healthy controls. Moreover, in the lesional skin of AD patients, higher S. aureus cell numbers significantly correlated with the higher total bacterial load. Furthermore, significantly more severe AD patients presented with higher S. aureus cell number and total bacterial load compared to patients with mild or moderate AD. Our results indicate that severe AD patients exhibit S. aureus driven increased bacterial skin colonization. Overall, bacterial quantification gives important insights in addition to microbiome composition by sequencing.

Mastitis oz. vnetje mlečne žleze je ena od gospodarsko najpomembnejših bolezni prežvekovalcev za prirejo mleka. Mastitis običajno povzročajo bakterije. Celične kulture lahko služijo kot alternativa študijam na živih živali in predstavljajo in vitro model za preučevanje poteka bolezni, kar je ključno za vzpostavitev uspešnih strategij za preprečevanje mastitisa. V eksperimentalnem delu diplomske naloge smo vzpostavili primarno celično kulturo epitelnih celic mlečne žleze goveda – pbMEC in jo uporabili za preučevanje imunskega odziva ob imunostimulaciji. pbMEC smo vzpostavili na neinvaziven način iz mleka in z imunofluorescenco potrdili prisotnost epitelnih celic. Celice smo imunostimulirali z virulentnim dejavnikom Gram negativnih bakterij LPS ter jih inkubirali 3 in 24 ur. Po pretekli inkubaciji smo izolirali RNA, jo reverzno prepisali v cDNA in z metodo qPCR določili diferenčno izražanje genov za citokina IL-8 in TNF-alfa. Rezultati so pokazali, da se je izražanje IL-8 in TNF-alfa po imunostimulaciji z LPS povečalo, kar kaže na to, da je kultura pbMEC imunokompetentna. Izražanje IL-8 je bilo po 24 urah višje kot po 3 urah, medtem ko je bilo izražanje TNF-alfa višje po 3 urah in je bilo po 24 urah le še nekoliko povečano. Rezultati so skladni z ugotovitvami prejšnjih študij ter kažejo na to, da je kultura pbMEC potencialno uporaben in vitro model za preučevanje imunskega odziva ob mastitisu. Mastitis, inflammation of the mammary gland, is one of the most economically important diseases of dairy ruminants. Mastitis is usually caused by bacteria. Cell cultures can serve as an alternative to live animal studies and provide an in vitro model to study the course of the disease, which is crucial for the establishment of successful mastitis prevention strategies. In the experimental part of the thesis, we established a primary cell culture of bovine mammary epithelial cells – pbMEC, and used it as a cell model to study mastitis. pbMEC were established in a non-invasive manner from milk, and the presence of epithelial cells was confirmed by immunofluorescence. The cells were immunostimulated with the Gram negative bacterial virulence factor LPS and incubated for 3 and 24 hours. After the incubation, RNA was isolated, reverse transcribed into cDNA and the differential expression of the IL-8 and TNF-alpha cytokines was determined by qPCR. The results showed that the expression of IL-8 and TNF-alpha increased after immunostimulation with LPS, suggesting that the pbMEC culture is immunocompetent. The expression of IL-8 was higher after 24 hours than after 3 hours, while the expression of TNF-alpha was higher after 3 hours and was only slightly elevated after 24 hours. The results are consistent with the findings of previous studies and suggest that pbMEC culture is a potentially useful in vitro model to study the immune response during mastitis.

Frontline laboratories are adopting culture-independent diagnostic testing (CIDT) such as nucleic acid amplification tests (NAATs) due to numerous advantages over culture-based testing methods. Paradoxically, the viability of pathogens, a crucial factor determining active infections, cannot be confirmed with current NAATs alone. A recent development of viability PCR (vPCR) was introduced to mitigate this limitation associated with real-time PCR (qPCR) by using a DNA-intercalating dye to remove residual and dead cell DNA. This study assessed the applicability of the vPCR assay on diarrheal stools. Eighty-five diarrheal stools confirmed for Salmonellosis were tested via qPCR and vPCR using in-house primers and probe targeting the invA gene. vPCR-negative stools (Ct cut off > 31) were enriched in mannitol selenite broth (MSB) to verify low bacterial loads. vPCR assay showed ~89% sensitivity (qPCR- and vPCR-positive stools: 76/85). vPCR-negative stools (9/85; qPCR-positive: 5; qPCR-negative: 4) were qPCR- and culture-positive post-MSB-enrichment and confirmed the presence of low viable bacterial loads. Random sampling error, low bacterial loads, and receiving stools in batches could contribute to false negatives. This is a pilot study and further investigations are warranted to explore vPCR to assess pathogen viability in a clinical setting, especially when culture-based testing is unavailable.

Since the introduction of live-attenuated rotavirus vaccines in Belgium in 2006, surveillance has routinely detected rotavirus vaccine-derived strains. However, their genomic landscape and potential role in gastroenteritis have not been thoroughly investigated. We compared VP7 and VP4 nucleotide sequences obtained from rotavirus surveillance with the Rotarix vaccine sequence. As a result, we identified 80 vaccine-derived strains in 5125 rotavirus-positive infants with gastroenteritis from 2007 to 2018. Using both viral metagenomics and reverse transcription qPCR, we evaluated the vaccine strains and screened for co-infecting enteropathogens. Among the 45 patients with known vaccination status, 39 were vaccinated and 87% received the vaccine less than a month before the gastroenteritis episode. Reconstruction of 30 near complete vaccine-derived genomes revealed 0-11 mutations per genome, with 88% of them being non-synonymous. This, in combination with several shared amino acid changes among strains, pointed at selection of minor variant(s) present in the vaccine. We also found that some of these substitutions were true revertants (e.g., F167L on VP4, and I45T on NSP4). Finally, co-infections with known (e.g., Clostridioides difficile and norovirus) and divergent or emerging (e.g., human parechovirus A1, salivirus A2) pathogens were detected, and we estimated that 35% of the infants likely had gastroenteritis due to a 'non-rotavirus' cause. Conversely, we could not rule out the vaccine-derived gastroenteritis in over half of the cases. Continued studies inspecting reversion to pathogenicity should monitor the long-time safety of live-attenuated rotavirus vaccines. All in all, the complementary approach with NGS and qPCR provided a better understanding of rotavirus vaccine strain evolution in the Belgian population and epidemiology of co-infecting enteropathogens in suspected rotavirus vaccine-derived gastroenteritis cases. ispartof: VACCINE vol:40 issue:35 pages:5114-5125 ispartof: location:Netherlands status: published

International audience; Anaerobic digestion (AD) is an attractive bioprocess for waste treatment and energy recovery through methane-rich biogas production. Under temperate to cold climate, the implementation of AD for low-organic load wastewater treatment has been limited to date, due to the energetic and economic cost of maintaining optimal mesophilic temperature. Hence, we aim at (i) exploring the biotechnological potential of a microbial inoculum from Antarctic soils and sediments to run AD at low temperatures; and (ii) evaluating the effect of temperature over a psychrophilic-mesophilic range on both methane production rates and microbial community composition. Methane production stimulated by acetate amendment was detected from 5 to 37 °C, with a maximum at 25 °C, corresponding to the highest relative abundance of methanogenic archaea (c. 21.4% of the total community). From 5 to 25 °C, the predominant methanogen was Methanosaeta, while it shifted to Methanocorpusculum at 30 °C. Compared with an industrial mesophilic sludge, the relative methane production rate at 5 °C (compared to the maximum) was 40% greater in the Antarctic inoculum. Microbial communities from permanently cold Antarctic sediments efficiently produce methane at low temperatures revealing a biotechnological potential for the treatment of low-organic load residues in cold regions.

Triazole fungicides are widely used to treat fungal pathogens in field crops, but very few studies have investigated whether fields of these crops constitute hotspots of azole resistance in Aspergillus fumigatus. Soil samples were collected from 22 fields in two regions of eastern France and screened for triazole residues and azole-resistant A. fumigatus (ARAf). Real-time quantitative PCR (qPCR) was used to quantify A. fumigatus in these soil samples. All the plots contained tebuconazole at concentrations from 5.5 to 19.1 ng/g of soil, and 5 of the 22 plots also contained epoxiconazole. Only a few fungal isolates were obtained, and no ARAf was detected. A. fumigatus qPCR showed that this fungal species was, on average, 5000 times more common in soil from flowerbeds containing ARAf than in soil from field crops. Thus, field-crop soils do not appear to promote A. fumigatus development, even if treated with azole fungicides, and cannot be considered hotspots of resistance. Indeed, our results suggest that they are instead a coldspot of resistance and highlight how little is known about the ecological niche of this species.

The European eel Anguilla anguilla is a critically endangered catadromous species. There is an urgent need for close surveillance of the populations that are still viable in European rivers. The species is difficult to observe in freshwater because of its bottom-dwelling behavior; the currently employed methods of eel monitoring in Europe based on the physical capture of individuals are stressful and may cause mortality. Here, we present a new highly sensitive method based on an A. anguilla-specific qPCR marker designed within the cytochrome oxidase I mitochondrial gene for application on environmental DNA (eDNA). Since the detectability of eDNA depends on the hydrographic conditions, we applied correction for altitude and a linear model and were able to predict the eel biomass from the eDNA in the different rivers of northern Spain still holding wild populations. The method was validated by electrofishing surveys. This novel eDNA-based marker allows for estimating the European eel biomass in running waters from small 1.5 L water samples and could complement, or replace in some cases, current eel surveys without disturbing wild populations.

Virgin olive oil (VOO), characterized by its unique aroma, flavor, and health benefits, is subject to adulteration with the addition of oils obtained from other edible species. The consumption of adulterated olive oil with nut species, such as hazelnut or almond, leads to health and safety issues for consumers, due to their high allergenic potential. To detect almond and hazelnut in olive oil, several amplification systems have been analyzed by qPCR assay with a SYBR Green post-PCR melting curve analysis. The systems selected were Cora1F2/R2 and Madl, targeting the genes coding the allergenic protein Cor a 1 (hazelnut) and Pru av 1 (almond), respectively. These primers revealed adequate specificity for each of the targeted species. In addition, the result obtained demonstrated that this methodology can be used to detect olive oil adulteration with up to 5% of hazelnut or almond oil by a single qPCR assay, and with a level as low as 2.5% by a nested-qPCR assay. Thus, the present research has shown that the SYBR-based qPCR assay can be a rapid, precise, and accurate method to detect adulteration in olive oil.

Modern integrated farming systems encourage the search for alternative (non-chemical), highly effective methods of plant protection. In this study, the fungistatic effect of fennel essential oil (FEO) and hop essential oil (HEO) on fungal growth and their ability to treat Fusarium wilt was investigated. The study was conducted in vitro and in pot experiments. The severity of infection was assessed by disease index (DI), presence of Fusarium culmorum gDNA (qPCR) and anatomical analyses of infected plant tissue using an optical (OM) and scanning electron microscope (SEM). Laboratory analyses showed that FEO inhibits mycelial growth of Fusarium fungi (F. avenaceum, F. culmorum, F. equiseti, F. oxysporum, F. poae, F. solani, F. sporotrichioides, F. tricinctum), Botrytis cinerea and Cylindrocarpon destructans more effectively than HEO. FEO at a concentration of 2000 ppm completely inhibited the growth of F. culmorum, F. poae and B. cinerea. Both essential oils reduced the severity of Fusarium wilt caused by F. culmorum in pea plants (DI, OM, SEM). The qPCR shows that both essential oils are also able to reduce the synthesis of trichothecenes in the tissues of infected pea plants. The results of the study suggest that FEO and HEO represent a broad spectrum bio-fungicidal agent that can be applied directly to plants at a concentration of 500 ppm, greatly reducing the level of infection.

Little information is presently available regarding the frequency of the silent shedders of respiratory viruses in healthy sport horses and their impact on environmental contamination. Therefore, the aim of this study was to investigate the detection frequency of selected respiratory pathogens in nasal secretions and environmental stall samples of sport horses attending a multi-week equestrian event during the summer months. Six out of fifteen tents were randomly selected for the study with approximately 20 horse/stall pairs being sampled on a weekly basis. Following weekly collection for a total of 11 weeks, all samples were tested for the presence of common respiratory pathogens (EIV, EHV-1, EHV-4, ERAV, ERBV, and Streptococcus equi ss equi (S. equi)) using qPCR. A total of 19/682 nasal swabs (2.8%) and 28/1288 environmental stall sponges (2.2%) tested qPCR-positive for common respiratory pathogens. ERBV was the most common respiratory virus (17 nasal swabs, 28 stall sponges) detected, followed by EHV-4 (1 nasal swab) and S. equi (1 nasal swab). EIV, EHV-1, EHV-4 and ERAV were not detected in any of the study horses or stalls. Only one horse and one stall tested qPCR-positive for ERBV on two consecutive weeks. All the other qPCR-positive sample results were related to individual time points. Furthermore, only one horse/stall pair tested qPCR-positive for ERBV at a single time point. The study results showed that in a selected population of sport horses attending a multi-week equestrian event in the summer, the frequency of the shedding of respiratory viruses was low and primarily restricted to ERBV with little evidence of active transmission and environmental contamination.

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture’s limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

Background: Terrein (Terr) is a bioactive marine secondary metabolite that possesses antiproliferative/cytotoxic properties by interrupting various molecular pathways. Gemcitabine (GCB) is an anticancer drug used to treat several types of tumors such as colorectal cancer; however, it suffers from tumor cell resistance, and therefore, treatment failure. Methods: The potential anticancer properties of terrein, its antiproliferative effects, and its chemomodulatory effects on GCB were assessed against various colorectal cancer cell lines (HCT-116, HT-29, and SW620) under normoxic and hypoxic (pO2 ≤ 1%) conditions. Further analysis via flow cytometry was carried out in addition to quantitative gene expression and 1HNMR metabolomic analysis. Results: In normoxia, the effect of the combination treatment (GCB + Terr) was synergistic in HCT-116 and SW620 cell lines. In HT-29, the effect was antagonistic when the cells were treated with (GCB + Terr) under both normoxic and hypoxic conditions. The combination treatment was found to induce apoptosis in HCT-116 and SW620. Metabolomic analysis revealed that the change in oxygen levels significantly affected extracellular amino acid metabolite profiling. Conclusions: Terrein influenced GCB’s anti-colorectal cancer properties which are reflected in different aspects such as cytotoxicity, cell cycle progression, apoptosis, autophagy, and intra-tumoral metabolism under normoxic and hypoxic conditions.

Background Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34. Results NSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNFα levels was seen in the infected neurons, which is of interest because TNFα protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron. Conclusions The findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.

16 pages, 2 figures, 1 table.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY), An increasing number of studies using marine environmental DNA (eDNA) approaches are showing its potential application in marine fisheries management by helping and simplifying some of the labor-intensive traditional surveys required to assess exploited populations and ecosystem status. eDNA approaches (i.e. metabarcoding and targeted) can support to ecosystem-based fisheries management by providing information on species composition; surveillance of invasive, rare and/or endangered species; and providing estimates of species abundance. Due to these potential uses in fisheries and conservation sciences, the number of studies applying eDNA approaches in marine habitats has expanded in the very last few years. However, a lack of consistency across studies when applying pipelines for data analyses, makes results difficult to compare among them. Such lack of consistency is partially caused by poor knowledge in the management of raw sequences data, and analytical methods allowing comparative results. Hence, we review here the essential steps of eDNA data processing and analyses to get sound, reproducible, and comparable results, providing a set of bioinformatics tools useful for each step. Altogether this review presents the state of the art of eDNA data analyses towards a comprehensive application in fisheries management promoting sustainability, The information and views set out in this manuscript are based on scientific data and information collected under Service Contract “Improving cost-efficiency of fisheries research surveys and fish stocks assessments using next-generation genetic sequencing methods [EMFF/2018/015]” signed with the European Climate, Infrastructure and Environment Executive Agency (CINEA) and funded by the European Union. NP-M is supported by MSCA-PF FishAdapt 101066785

Staphylococcus spp. have been associated with cases of healthcare associated infections due to their high incidence in isolates from the hospital environment and their ability to cause infections in immunocompromised patients; synthesize biofilms on medical instruments, in the case of negative coagulase species; and change in genetic material, thus making it possible to disseminate genes that code for the acquisition of resistance mechanisms against the action of antibiotics. This study evaluated the presence of blaZ, femA, and mecA chromosomal and plasmid genes of Staphylococcus spp. using the qPCR technique. The results were associated with the phenotypic expression of resistance to oxacillin and penicillin G. We found that the chromosomal femA gene was present in a greater proportion in S. intermedius when compared with the other species analyzed, while the plasmid-borne mecA gene was prevalent in the S. aureus samples. The binary logistic regression performed to verify the association among the expression of the genes analyzed and the acquisition of resistance to oxacillin and penicillin G were not significant in any of the analyses, p > 0.05.

Background: This study aimed to describe the kinetics of Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients. Methods: Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up. Results: A significant reduction in the parasite load was observed after treatment (P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six. Conclusions: We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases.

In Brazil, blood donation is regulated by the Brazilian Ministry of Health, and all States follow the same protocol for clinical and laboratory screening. Brazil is an endemic country for Chagas disease (CD), caused by Trypanosoma cruzi, and for leishmaniasis, caused by a species of Leishmania spp. Screening for leishmaniosis is not routinely performed by blood banks. Given the antigenic similarity between T. cruzi and Leishmania spp., cross-reactions in serological tests can occur, and inconclusive results for CD have been found. The objective of this study was to apply molecular techniques, e.g., nPCR, PCR, and qPCR, to clarify cases of blood donation candidates with non-negative serology for CD and to analyze the difference between the melting temperature during real-time PCR using SYBR Green. Thirty-seven cases that showed non-negative results for CD using chemiluminescent microparticle immunoassay (CMIA) tests from blood banks in Campo Grande, MS, and Campinas, SP, were analyzed. In the serum samples, 35 samples were evaluated by ELISA, and 24.3% (9/35) showed positive results for CD. nPCR was able to detect 12 positive results in 35 samples (34.28%). qPCR for T. cruzi was quantifiable in the samples that showed a value ≥0.002 par eq/mL (parasite equivalents per milliliter), and in 35 samples, 11 (31.42%) were positive. Of all evaluated samples using the described tests (CMIA, ELISA, nPCR, and qPCR), 18 (48.6%) were positive for CD. For MCA by qPCR, the melting temperature was 82.06 °C ± 0.46 for T. cruzi and 81.9 °C ± 0.24 for Leishmania infantum. The Mann–Whitney test showed a significant value of p < 0.0001. However, the differentiation between T. cruzi and L. infantum could not be considered due to temperature overlap. For leishmaniasis, of the 35 samples with non-negative serology for CD tested by the indirect fluorescent antibody test (IFAT), only one sample (2.85%) was positive (1:80). The PCR for Leishmania spp. was performed on 36 blood samples from donation candidates, and all were negative. qPCR for L. infantum showed 37 negative results for the 37 analyzed samples. The data presented here show the importance of performing two different tests in CD screening at blood banks. Molecular tests should be used for confirmation, thereby improving the blood donation system.

Problematic fermentations frequently drive economic losses and logistic problems in the winemaking industry. Previous studies have determined thermal conditions leading to problematic fermentations, selecting two contrasting yeast strains for further transcriptomic analysis. Saccharomyces cerevisiae SBB11 showed strong thermosensitivity towards heat shock, while S. cerevisiae PDM was found to be thermotolerant. The aim of this study was to select genes with significantly upregulated expression to be later used as biomarkers for early detection of sluggish fermentation associated with heat shock. Candidate genes were selected from previously obtained RNA-seq data. Alcoholic fermentations were conducted with 4 S. cerevisiae strains SBB11, PDM, M2 and ICV D21. Heat shocks on day 3 of alcoholic fermentation were applied at 36 and 40 °C for 16 h. S. cerevisiae cells were collected at different times after heat shock onset for qPCR analysis of candidate gene expression over time. Three genes showed promising results; SSA1, MGA1 and OPI10 significantly increased expression with respect to the control. The selected genes showed increased expression during the first 9 h post heat shock and are proposed for early detection of sluggish fermentations associated with heat shock., This research was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) through the projects PIP 2014-2016 (Ref 11220130100694) (Argentina), PICT 2016-3406 FONCyT (Argentina) and PICT 2020-2260 FONCYT (Argentina); Instituto Nacional de Tecnología Agropecuaria INTA-PD-E6-I116; and Consejo Superior de Investigaciones Científicas (CSIC) I-COOP B 20316 (Spain).

Streptococcus pneumoniae causes significant morbidity and mortality among older adults. Detection of pneumococcal carriage is an accepted endpoint in pneumococcal conjugate vaccine studies. However, low sensitivity of culture-based approaches and nasopharyngeal samples have hampered adult S. pneumoniae carriage studies in the past. In contrast, detection of adult S. pneumoniae carriers with qPCR-based approaches can achieve high sensitivity and specificity and qPCR-based testing of oral samples improves accuracy of adult carriage detection. In this Viewpoint we outline a strategy for accurate qPCR-based testing. We recommend a dual-target approach for S. pneumoniae qPCR detection as no genetic target is universally present among or solely unique to it. Furthermore, we advise the evaluation of concordance among quantified qPCR targets to improve the accuracy of S. pneumoniae testing and qPCR-based serotyping. We do not recommend omission of qPCR-based oral sample testing as it will likely result in an underestimation of true adult carrier rates.

Streptococcus pneumoniae causes significant morbidity and mortality among older adults. Detection of pneumococcal carriage is an accepted endpoint in pneumococcal conjugate vaccine studies. However, low sensitivity of culture-based approaches and nasopharyngeal samples have hampered adult S. pneumoniae carriage studies in the past. In contrast, detection of adult S. pneumoniae carriers with qPCR-based approaches can achieve high sensitivity and specificity and qPCR-based testing of oral samples improves accuracy of adult carriage detection. In this Viewpoint we outline a strategy for accurate qPCR-based testing. We recommend a dual-target approach for S. pneumoniae qPCR detection as no genetic target is universally present among or solely unique to it. Furthermore, we advise the evaluation of concordance among quantified qPCR targets to improve the accuracy of S. pneumoniae testing and qPCR-based serotyping. We do not recommend omission of qPCR-based oral sample testing as it will likely result in an underestimation of true adult carrier rates.

Xylella fastidiosa (Xf) is a Gram-negative plant bacterium that causes severe diseases affecting several economically important crops in many countries. To achieve early detection of the pathogen, a droplet digital PCR (ddPCR)-based approach was used to detect the bacterium at low concentrations in different plant species and insect vectors. In this study, we implemented the reaction conditions of a previously developed ddPCR assay, and we validated its use to detect Xf in insect vectors as well as in a broader list of host species. More specifically, the sensitivity and accuracy of the protocol were assessed by testing five plant matrices (Olea europaea, Nerium oleander, Vitis vinifera, Citrus sinensis, and Prunus dulcis), and for the first time, the insect vector (Philaenus spumarius), was either naturally infected or artificially spiked with bacterial suspension at known concentrations. The lowest concentrations detected by ddPCR were 5 ag/µL of bacterial DNA and 1.00 × 102 CFU/mL of bacterial cells. Both techniques showed a high degree of linearity, with R2 values ranging from 0.9905 to 0.9995 and from 0.9726 to 0.9977, respectively, for qPCR and ddPCR. Under our conditions, ddPCR showed greater analytical sensitivity than qPCR for O. europea, C. sinensis, and N. oleander. Overall, the results demonstrated that the validated ddPCR assay enables the absolute quantification of Xf target sequences with high accuracy compared with the qPCR assay, and can support experimental research programs and the official controls, particularly when doubtful or inconclusive results are recorded by qPCR.

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756–0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872–1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.

Asociacijske študije v celotnem genomu (GWAS) so študije, ki obravnavajo številne pogoste genetske različice pri različnih osebkih, da bi ugotovili potencialne povezave z boleznimi. Ko so odkrite nove genetske povezave, lahko raziskovalci informacije uporabijo za razvoj boljših strategij za odkrivanje, zdravljenje in preprečevanje bolezni. GWAS študije so do zdaj podale veliko genskih lokusov povezanih s kostnimi boleznimi. Eden izmed odkritih lokusov je enonukleotidni polimorfizem rs7851693 v intronu gena za FUBP3, ki je povezan s tveganjem za zlome kosti oz. osteoporozo. Vloga proteina FUBP3 pri metabolizmu kosti še ni znana. V želji, da bi prispevali k raziskavam, smo poskušali določiti povezavo med izražanjem gena FUBP3 in kostnim rakom (osteosarkomom). V okviru magistrske naloge, smo najprej poskušali ugotoviti ali gen FUBP3 igra vlogo pri tvorbi kosti z uporabo tehnike popolnega izbitja gena med osteogeno diferenciacijo človeških osteosarkomskih celic (HOS, angl. human osteosarcoma cells ). Potek mineralizacije smo paralelno primerjali s potekom mineralizacije pri divjem tipu HOS celic. Z metodo barvanja Alizarin Red S in končnim kvantitativnim vrednotenjem smo ugotovili, da mineralizacija hitreje poteka pri divjem tipu, kar nakazuje, da gen FUBP3 ima določeno vlogo pri tvorbi kosti. Hitrejša mineralizacija je bila vidna tudi pod mikroskopom. Poleg tega smo naredili transfekcijo človeških HOS celic, z izbitim FUBP3 genom, s proteinom FUBP3 ribe zebrice (zfFUBP3) in praznim vektorjem (pCMV), da bi preverili ali povišano izražanje zfFUBP3 vpliva na osteogeno diferenciacijo. Rezultate smo tudi kvantitativno ovrednotili in ugotovili, da ima zfFUBP3 vpliv na tvorbo kosti in lahko sklepamo, da igra podobno vlogo kot človeški FUBP3. Poleg opisanih eksperimentov, smo z metodo PCR v realnem času (qPCR) kvantitativno izmerili še izražanje treh osteoblastnih genov – kolagena tipa I (COL1A1), osteokalcina (OC) in transkripcijskega faktora II (RUNX2). Rezultati magistrske naloge kažejo, da igra protein FUBP3 pomembno vlogo pri mineralizaciji osteosarkomske celične linije HOS. Genome-wide association studies (GWAS) are studies that look at many common genetic variants in different individuals to identify potential associations with diseases. When new genetic associations are identified, researchers can use the information to develop better strategies for detecting, treating and preventing diseases. GWAS studies have so far identified many gene loci associated with bone diseases. One of the discovered loci is the single – nucleotide polymorphism rs7851693 in the intron of the FUBP3 gene, which is associated with the risk of bone fractures and osteoporosis. The role of the FUBP3 protein in bone metabolism is not yet known. In order to contribute to research, we tried to determine the relationship between the expression of the FUBP3 gene and bone cancer (osteosarcoma). As a part of the master’s thesis, we tried to determine whether the FUBP3 gene plays a role in bone formation using the technique of complete gene knockout during osteogenic differentiation of human osteosarcoma cells (HOS). The course of mineralization was compared in parallel with the course of mineralization in wild-type HOS cells. Using the Alizarin Red S staining method and the final quantitative evaluation, we found out that mineralization occurs faster in the wild-type cells, which suggests the FUBP3 gene has a certain role in bone formation. Faster mineralization was also visible under the microscope. In addition, we transfected human FUBP3 gene knocked out cells, with zfFUBP3 (zebrafish FUBP3) and empty plasmid, in order to check whether increased expression of this protein has an impact on osteogenic differentiation. We also evaluated the results quantitatively and found that zfFUBP3 has an impact in bone formation and we can conclude that it plays a similar role to human FUBP3. In addition to the described experiments, we quantitatively measured the expression of three more genes – collagen type I (COL1A1), osteocalcin (OC) and transcription factor II (RUNX2) – using the real-time PCR (qPCR) method. The results of the master's thesis show that the protein FUBP3 plays an important role in the mineralization of the osteosarcoma cell line HOS.

Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples—or in this case, chicken droppings.

This paper focuses on the comparison of microbial biomass increase (cell culture growth) using field-relevant testing methods and moving away from colony counts. Challenges exist in exploring the antimicrobial growth of fastidious strains, poorly culturable bacteria and bacterial communities of environmental interest. Thus, various approaches have been explored to follow bacterial growth that can be efficient surrogates for classical optical density or colony-forming unit measurements. Here, six species grown in pure culture were monitored using optical density, ATP assays, DNA concentrations and 16S rRNA qPCR. Each of these methods have different advantages and disadvantages concerning the measurement of growth and activity in complex field samples. The species used as model systems for monitoring were: Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. All four techniques were found to successfully measure and detect cell biomass/activity differences, though the shape and accuracy of each technique varied between species. DNA concentrations were found to correlate the best with the other three assays (ATP, DNA concentrations and 16S rRNA-targeted qPCR) and provide the advantages of rapid extraction, consistency between replicates and the potential for downstream analysis. DNA concentrations were determined to be the best universal monitoring method for complex environmental samples.

Suitable reference genes are one of the necessary conditions for obtaining reliable results by real-time fluorescence quantitative PCR (qPCR). In this study, the expression of the 10 common candidate reference genes (18s rRNA, rps27, cnbp, rpl7, ube2, hsp-at, gapdh, β-actin, rpl13a, 1-ef1a) at different developmental stages of gonad and embryo of Takifugu bimaculatus were analyzed by qPCR. And the expression stability of these reference genes is analyzed by GeNorm, NormFinder and Bestkeeper softwares. The results showed that the expression stability of 1-ef1a was the highest (p

Striga hermonthica (Del.) Benth is a devastating parasitic weed in Sub-Saharan Africa (SSA) and its soil seedbank is the major factor contributing to its prevalence and persistence. To date, there is a little information on the Striga seedbank density in agricultural fields in SSA due to the lack of reliable detection and quantification methods. We developed a high-throughput method that combines density- and size-based separation techniques with quantitative polymerase chain reaction (qPCR)-based detection of Striga seeds in soil. The method was optimised and validated by introducing increasing numbers of Striga seeds in two physicochemically different Striga-free agricultural soils. The results showed that as little as one seed of S. hermonthica per 150 g of soil could be detected. This technique was subsequently tested on soil samples of 48 sorghum fields from different agro-ecological zones in Ethiopia to map the geospatial distribution of the Striga seedbank along a trajectory of more than 1500 km. Considerable variation in Striga seed densities was observed. Striga seeds were detectable in 75% of the field soils with densities up to 86 seeds per 150 g of soil. The Striga seed density in soil and the number of emerged Striga plants in the field showed a non-linear relationship. In conclusion, the method developed allows for accurate mapping of the Striga seedbank in physicochemically diverse SSA field soils and can be used to assess the impact of management strategies on Striga seedbank dynamics.

International audience; Quantitative resistance (QR) to Leptosphaeria maculans in Brassica napus has proven to be very useful for breeding resistant varieties with increased potential for durability. To date, however, QR has been assessed primarily in the field after a long plant developmental cycle and its expression is influenced by environmental conditions. An evaluation method in controlled conditions would offer the possibility to evaluate the level of QR in a shorter time and to study the kinetics of fungal growth in the stem in relation to susceptibility/resistance levels conferred by quantitative trait loci (QTL). We used a cut-petiole inoculation method coupled with both visual and PCR-based phenotyping at different time-points after inoculation, which revealed different kinetics of symptom development and pathogen growth among varieties. The results obtained with this method were partly consistent with field resistance phenotyping data, and allowed detection of the effect of at least two resistance QTL alleles introduced in near-isogenic lines. This method would be useful to compare a wider range of host varieties, to study the spectrum effect of resistance QTL or to decipher the mechanisms underlying this quantitative resistance.

Felid alphaherpesvirus type 1 (FHV-1) is an important cause of respiratory and ocular diseases in cats worldwide. Mice have been widely used to study the pathogenesis of several human and animal viruses, especially herpesviruses. This study aimed to verify whether BALB/c mice are susceptible to FHV-1 infection. The animals were intranasally inoculated with FHV-1 and their clinical signs were observed from 3 days post-infection (dpi). At 10 dpi, the animals were euthanized and the lungs, liver, spleen, and kidneys were collected for histopathological examination and quantitative polymerase chain reaction. The results showed that mice were infected with FHV-1 and reproduced several features of the disease observed in its natural host. Histological lesions and viral DNA were found in all sampled tissues, with a higher frequency of FHV-1 DNA copies detected in the lungs. All mice were seroconverted to FHV-1 at 7 dpi. To our knowledge, this is the first report of experimental infection of BALB/c mice with FHV-1. Our findings demonstrate that this murine model can contribute to our understanding of FHV-1 pathogenesis and may be useful for trials against this virus. O herpesvírus felino tipo 1 (FHV-1) é um importante agente causador de doença respiratória e ocular em gatos em todo o mundo. Camundongos têm sido amplamente utilizados para estudar a patogenia de diversos vírus humanos e animais, especialmente os herpesvírus. O objetivo deste estudo foi verificar se camundongos BALB/c poderiam ser suscetíveis a infecção pelo FHV-1. Os animais foram inoculados com FHV-1 intranasalmente e sinais clínicos foram observados a partir do dia 3 após a infecção (dpi). Após dez dias da inoculação, os animais foram sacrificados e os pulmões, fígado, baço e rins foram coletados para exame histopatológico e PCR quantitativo (qPCR). Os resultados mostraram que os camundongos foram infectados com o FHV-1 e reproduziram várias características da doença que são observadas em seu hospedeiro natural. Lesões histológicas e DNA viral foram detectadas em todos os tecidos amostrados, com maior frequência de DNA do FHV-1 nos pulmões. Todos os camundongos soroconverteram para FHV-1 aos 7 dpi. Para nosso conhecimento, este estudo é o primeiro relato de infecção experimental de camundongos BALB/c com FHV-1. Nossos resultados demonstraram que esse camundongo pode contribuir paro entendimento da patogenia do FHV-1 e pode ser um modelo útil para ensaios contra esse agente.