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Rosa damascena Mill., commonly known as the King Flower, is a fragrant and important species of the Rosaceae family. It is widely used in the perfumery and pharmaceutical industries. The scent and color of the flowers are significant characteristics of this ornamental plant. This study aimed to investigate the relative expression of MYB1, CCD1, FLS, PAL, CER1, GT1, ANS and PAR genes under two growth stages (S1 and S2) in two morphs. The CCD1 gene pathway is highly correlated with the biosynthesis of volatile compounds. The results showed that the overexpression of MYB1, one of the important transcription factors in the production of fragrance and color, in the Hot pink morph of sample S2 increased the expression of PAR, PAL, FLS, RhGT1, CCD1, ANS, CER1, and GGPPS. The methyl jasmonate (MeJA) stimulant had a positive and cumulative effect on gene expression in most genes, such as FLS in ACC.26 of the S2 sample, RhGT1, MYB1, CCD1, PAR, ANS, CER1, and PAL in ACC.1. To further study, a comprehensive analysis was performed to evaluate the relationship between the principal volatile compounds and colors. Our data suggest that the rose with pink flowers had a higher accumulation content of flavonoids and anthocyanin. To separate essential oil compounds, GC/MS analysis identified 26 compounds in four samples. The highest amount of geraniol, one of the main components of damask rose, was found in the Hot pink flower, 23.54%, under the influence of the MeJA hormone.
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Flores , Regulación de la Expresión Génica de las Plantas , Odorantes , Rosa , Rosa/genética , Rosa/metabolismo , Flores/genética , Flores/metabolismo , Odorantes/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacología , Compuestos Orgánicos Volátiles/metabolismo , Genes de Plantas , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Pigmentación/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Acetatos/farmacología , Acetatos/metabolismo , ColorRESUMEN
Type III secretion system (T3SS) is an important virulence system in Gram-negative bacteria. In this investigation, different environmental conditions that regulate the expression of T3SS genes in Yersinia ruckeri were investigated aimed at obtaining a better understanding about its modulation after various environmental challenges. Four isolates of Y. ruckeri CSF007-82, ATCC29473, A7959-11 and YRNC10 were cultivated under the diverse in vitro challenges iron depletion, high salt, low pH and in the presence of fish serum or in the fish cell culture (Chinook Salmon Embryo - CHSE). The transcriptional modulation of the chromosomal genes ysaV, ysaC, ysaJ and prgH of ysa were investigated using quantitative real-time PCR. The expression of prgH, ysaV, ysaC and ysaJ was differentially expressed in all four strains under evaluation. The highest gene expression levels were observed for Y. ruckeri YRNC10 AN after addition of 0.3 M NaCl in Luria Bertani broth. The results obtained from this study provide initial insights into T3SS responses in Y. ruckeri, which pave the way for further studies aimed at expanding our knowledge on the functional roles of the T3SS genes in Y. ruckeri.
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This study aims to investigate the relationship between toxoplasmosis and this pathway, which may be effective in the formation of epilepsy by acting through the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy. In the study, four different experimental groups were formed by selecting Toxoplasma gondii IgG positive and negative patients with idiopathic epilepsy and healthy controls. Experimental groups were as follows: Group 1: Epilepsy+/Toxo- (E+, T-) (n = 10), Group 2: Epilepsy-/Toxo- (E-, T-) (n = 10), Group 3: Epilepsy-/Toxo+ (E-, T+) (n = 10), Group 4: Epilepsy+/Toxo+ (E+, T+) (n = 10). HMGB1, RAGE, TLR4, TLR1, TLR2, TLR3, IRAK1, IRAK2, IKBKB, IKBKG, BCL3, IL1ß, IL10, 1 L8 and TNFα mRNA expression levels in the HMGB/RAGE/TLR4/NF-κB signalling pathway were determined by quantitative simultaneous PCR (qRT-PCR) after collecting blood samples from all patients in the groups. Statistical analysis was performed by one-way ANOVA followed by LSD post-hoc tests, and p < 0.05 was considered to denote statistical significance. The gene expression levels of HMGB1, TLR4, IL10, IL1B, IL8, and TLR2 were significantly higher in the G1 group than in the other groups (p < 0.05). In the G3 group, RAGE and BCL3 gene expression levels were significantly higher than in the other groups (p < 0.05). In the G4 group, however, IRAK2, IKBKB, and IKBKG gene expression levels were significantly higher than in the other groups (p < 0.05). HMGB1, TLR4, IRAK2, IKBKB, IL10, IL1B, IL1B, and IL8 in this signalling pathway are highly expressed in epilepsy patients in G1 and seizures occur with the stimulation of excitatory mechanisms by acting through this pathway. The signalling pathway in epilepsy may be activated by HMGB1, TLR4, and TLR2, which are considered to increase the level of proinflammatory cytokines. In T. gondii, this pathway is activated by RAGE and BCL3.
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Epilepsia , Proteína HMGB1 , FN-kappa B , Transducción de Señal , Receptor Toll-Like 4 , Toxoplasmosis , Humanos , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Proteína HMGB1/metabolismo , Proteína HMGB1/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Masculino , Femenino , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/parasitología , Adulto , Toxoplasmosis/parasitología , Toxoplasmosis/metabolismo , Toxoplasmosis/complicaciones , Toxoplasmosis/sangre , Toxoplasmosis/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Receptor para Productos Finales de Glicación Avanzada/genética , Estudios de Casos y Controles , Adulto Joven , Persona de Mediana Edad , Antígenos de Neoplasias , Proteínas Quinasas Activadas por MitógenosRESUMEN
Acanthamoeba spp., are ubiquitous protist which belongs to Free-Living Amoeba (FLA) group, is considered as causal agent of side-threatening keratitis or fatal encephalitis among other human infections. Besides, this parasite has been reported as host for other microorganisms important to human health such as Campylobacter spp. or Vibrio spp. among others. This role of Acanthamoeba as pathogen and environmental phagocyte has increased the reports confirming its presence in human related environments, acting as a water quality indicator. Considering the tide relationship between water and kitchen environments, and the high prevalence of Acanthamoeba in water sources, the present study aims to establish a quick and accurate protocol based on DNA extraction and a real time qPCR assay to detect Acanthamoeba spp. in dishcloths. The procedure has been validated by processing 17 used dishcloths. Our findings demonstrated the high sensitivity of the qPCR assay used which was capable of detecting up to one Acanthamoeba from an in vitro contaminated dishcloth. The protocol accurately detected 64.7% of positive samples for Acanthamoeba spp, (in 4 samples DNA concentrations corresponded to 1-102 amoebae). Our findings demonstrate the importance of FLA surveillance by efficient and sensitive methods since one amoeba is capable of colonizing human related food environments such as kitchens sinks and could be a potential source of infection.
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Acanthamoeba , Reacción en Cadena en Tiempo Real de la Polimerasa , Acanthamoeba/aislamiento & purificación , Acanthamoeba/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN Protozoario/genética , ADN Protozoario/análisis , Humanos , Sensibilidad y EspecificidadRESUMEN
Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.
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In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.
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Cinnamomum , Regulación de la Expresión Génica de las Plantas , Cloruro de Sodio , Cinnamomum/genética , Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de ReferenciaRESUMEN
【Objective】 To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR). 【Methods】 Based on the polymorphism of high-resolution level KIR alleles in Chinese population and the IPD-KIR database, KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR. Meanwhile, one negative control and one positive control specific amplifying human growth hormone (HGH) gene fragment were set to monitor the false positive and false negative results in PCR amplification, respectively. A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by authors. 【Results】 The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results, but two samples showed inconsistent results. One sample was negative for 2DS5 by Q-PCR but positive by PCR-SSP, another sample was positive for 2DS1 by Q-PCR but negative by PCR-SSP. The two doubtful samples were genotyped by sequencing-based typing (PCR-SBT) for 2DS5 and 2DS1, respectively. PCR-SBT results confirmed that the results of Q-PCR test was correct. 【Conclusion】 The KIR Q-PCR method established in this paper can provide accurate and reliable results for testing the presence or absence of KIR genes.
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BACKGROUND: Endometriosis characterized with existence of endometrial-like tissue outside the uterus. Fibrosis of ectopic lesions is an important feature of endometriosis. IL-4 induces fibrosis via fibroblast proliferation, collagen production and myofibroblast differentiation. Increasing of miR-21 expression promotes fibroblast activation and fibrosis expansion. The aim of study was to evaluate the expression of miR-21 and its relationship with IL-4 gene expression in endometrial ectopic and eutopic tissues of endometriosis patients. METHODS AND RESULTS: Ectopic and eutopic tissue samples were taken from 20 women with endometriosis, and control samples were taken from the endometrium of 20 endometriosis-free women. The relative expression of IL-4 and miR-21 evaluated by Real Time PCR. IL-4 relative gene expression was significantly increased in ectopic tissue compared to eutopic (p = 0.025) and control tissue (p = 0.021). The relative expression of miR-21 gene in ectopic tissue was increased compared to eutopic (p = 0.850) and control tissue (p = 0.978) but these differences were not significant. Also, the correlation between IL-4 and miR-21 relative gene expression was not significant (p = 0.083). CONCLUSION: The increased expression of miR-21 in endometrium of women with endometriosis may upregulate the IL-4 gene expression and lead to fibrosis. Further studies may suggest miR-21 and IL-4 as candidates for diagnosis of endometriosis.
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Coristoma , Endometriosis , MicroARNs , Humanos , Femenino , Endometriosis/genética , Endometriosis/metabolismo , Endometriosis/patología , Interleucina-4/genética , Coristoma/metabolismo , Coristoma/patología , Endometrio/metabolismo , Fibrosis , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Pesticides enter the environment through runoff and leaching and this raises public concern about effects on non-target organisms. Imidacloprid (IMI) a synthetic pesticide, has an unstable half-life, metabolized in minutes to weeks in the water. To evaluate the effects of IMI on the zebrafish liver, we conducted proteomic, molecular and biochemical analysis in a multi-level approach, to highlight the complementary features regarding the results of each method. Adult zebrafish were exposed to 60 mg/L IMI for 48 h and were evaluated using nLC-MS/MS for proteins, q-PCR analysis for expression of cat, gpx, pxr, ache, along with CAT and AChE enzyme activities and GSH and MDA assays. Based on proteomics, the regulation of antioxidant and immune responses, as well as gene transcription were significant processes affected. Apoptosis and ER stress pathways were upregulated and there was a down-regulation of cat and gpx genes. There was also elevated CAT activity and GSH and decreased MDA. Additionally, elevated AChE activity and up regulation of ache expression was observed. The multi-approach results included regulators of antioxidant, xenobiotic response and neuro-protective related proteins (genes and enzymes), which overall reflected harmful effects of IMI. Consequently, this study highlights the effects of IMI on zebrafish liver and reveals new potential biomarkers. In this respect, evaluated outcomes reveal the complementary features emphasizing the importance of studying chemicals using several methods. Our study provides deeper insights for future work in ecotoxicological studies regarding IMI and contribute to existing toxicity literature.
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Plaguicidas , Contaminantes Químicos del Agua , Animales , Plaguicidas/toxicidad , Plaguicidas/análisis , Plaguicidas/metabolismo , Antioxidantes/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Espectrometría de Masas en Tándem , Proteómica , Neonicotinoides , Nitrocompuestos/toxicidad , Hígado/metabolismo , Reacción en Cadena de la Polimerasa , Estrés Oxidativo , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/metabolismoRESUMEN
Toxoplasma gondii (T. gondii) is a parasite that obtains the iron it needs for its own metabolism from the host-cell iron pool. In this work, we aimed to investigate if iron supplementation or deficiency affected the course of T. gondii infection. Eighty mice were divided into four groups, each with 20 animals: Group (I): Uninfected control group. Group (II): Infected control group: injected with Phosphate buffered saline. Group (III): Infected group: received iron sucrose treatment. Group (IV): Infected group: treated with deferoxamine. Quantitative PCR studies were performed on days 3 and 8 post-infection to detect the expression of iron metabolism genes (hamp and ferroprotin) and immune-histochemical analysis to study the percentage of TNF-α and TGF-ß tissue expression. Iron supplementation induced progressions of infection evident by increased tissue expression of pro-inflammatory cytokine TNF-α and downregulation of TGF-ß which is mostly linked to suppression of the inflammatory process caused by T. gondii. Increased expression of TGF-ß and decreased expression of TNF-α was noticed when iron deprivation occurred. On day 3, we noticed increased expression in the hamp gene with iron supplementation while it decreases when the iron supply is low. On the contrary, iron deficiency increased ferroprotin gene expression whereas supplementing decreased it. On day 8, the level of expression of these genes returned to normal levels. These observations document the potential role of iron in controlling toxoplasmosis infection and indicate that the transcription of hamp and ferroprotin in T. gondii-infected cells appears to be regulated by a sophisticated indirect mechanism.
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Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA. Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures. Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than â¼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with â¼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris ), â¼27.7% in chromatin and small EVs and â¼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (â¼18.4%), and exosomes or protein complexes (â¼5.9%). Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.
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OBJECTIVES: To compare efficacy of aloe vera and probiotic mouthwashes vs fluoride mouthwash on Streptococcus mutans (S. mutans) in the plaque of orthodontic patients and to assess patient-reported outcomes and compliance. MATERIALS AND METHODS: This prospective randomized clinical trial included 90 patients aged 12-35 years and in permanent dentition, who were randomly allocated in a 1:1:1 ratio to three mouthwash groups: aloe vera, probiotic, or fluoride. Smartphone-based applications were used to improve patient compliance. The primary outcome was the change in S. mutans levels in plaque between two times: pre-intervention and after 30 days using real-time polymerase chain reaction (Q-PCR). Secondary outcomes were the evaluation of patient-reported outcomes and compliance. RESULTS: Mean differences between aloe vera vs probiotic: -0.53 (95% CI: -3.57 to 2.51), aloe vera vs fluoride: -1.99 (95% CI, -4.8 to 0.82), and probiotic vs fluoride: -1.46 (95% CI: -4.74 to 1.82) were not significant, P = .467. Intragroup comparison demonstrated a significant mean difference in all three groups of -0.67 (95% CI: -0.79 to -0.55), -1.27 (95% CI: -1.57 to -0.97), and -2.23 (95% CI: -2.44 to -2.00) respectively, P < .001. Adherence was above 95% in all groups. No significant differences in frequency of responses to patient-reported outcomes were found among groups. CONCLUSIONS: No significant difference in efficacy among the three mouthwashes in reducing S. mutans level in plaque was found. Patient-reported assessments concerning burning sensation, taste, and tooth staining found no significant differences among mouthwashes. Smartphone-based applications can help improve patient compliance.
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Aloe , Placa Dental , Probióticos , Humanos , Antisépticos Bucales/uso terapéutico , Fluoruros , Streptococcus mutans , Estudios Prospectivos , Probióticos/uso terapéuticoRESUMEN
Oxybenzone (BP-3) is an ultraviolet (UV) filter widely used in industries that is directly or indirectly released into the aquatic environment. However, little is known about its effects on brain performance. Here, we investigated whether BP-3 exposure affects the redox imbalance in zebrafish and how they respond to a task that requires memory of an aversive situation. Fish were exposed to BP-3 10 and 50 µg L-1 for 15 days and then tested using an associative learning protocol with electric shock as a stimulus. Brains were extracted for reactive oxygen species (ROS) measurement and qPCR analysis of antioxidant enzyme genes. ROS production increased for exposed animals, and catalase (cat) and superoxide dismutase 2 (sod 2) were upregulated. Furthermore, learning and memory were reduced in zebrafish exposed to BP-3. These results suggested that BP-3 may lead to a redox status imbalance, causing impaired cognition and reinforcing the need to replace the toxic UV filters with filters that minimize environmental effects.
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Benzofenonas , Contaminantes Químicos del Agua , Pez Cebra , Animales , Encéfalo/metabolismo , Catalasa/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno , Superóxido Dismutasa/metabolismo , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/análisis , Pez Cebra/metabolismo , Benzofenonas/toxicidadRESUMEN
Background: The SARS-CoV-2 gold standard detection method is an RT-qPCR with a previous step of viral RNA extraction from the patient sample either by using commercial automatized or manual extraction kits. This RNA extraction step is expensive and time demanding. Objective: The aim of our study was to evaluate the clinical performance of a simple SARS-CoV-2 detection protocol based on a fast and intense sample homogenization followed by direct RT-qPCR. Results: 388 nasopharyngeal swabs were analyzed in this study. 222 of them tested positive for SARS-CoV-2 by the gold standard RNA extraction and RT-qPCR method, while 166 tested negative. 197 of those 222 positive samples were also positive for the homogenization protocol, yielding a sensitivity of 88.74% (95% IC; 83.83 - 92.58). 166 of those negative samples were also negative for the homogenization protocol, so the specificity obtained was 97% (95% IC; 93.11 - 99.01). For Ct values below 30, meaning a viral load of 103 copies/uL, only 4 SARS-CoV-2 positive samples failed for the RNA extraction free method; for that limit of detection, the homogenizer-based method had a sensitivity of 97.92% (95% CI; 96.01 - 99.83). Conclusions: Our results show that this fast and cheap homogenization method for the SARS-CoV-2 detection by RT-qPCR is a reliable alternative of high sensitivity for potentially infectious SARS-CoV-2 positive patients. This RNA extraction free protocol would help to reduce diagnosis time and cost, and to overcome the RNA extraction kits shortage experienced during COVID-19 pandemic.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Prueba de COVID-19 , Pandemias , ARN Viral/genética , Sensibilidad y EspecificidadRESUMEN
Nitrogen (N) fertilizer application is an essential part of agricultural production in order to improve rice yields. However, long-term irrational application and low utilization of N fertilizer have caused a series of environmental problems. Biofertilizer is considered an effective alternative to N fertilizer. In this study, the effect of biofertilizer made of diazotrophic bacteria Bacillus sp. T28 combined with sea buckthorn pomace on the soil N changes and microbial community structure was conducted. Compared to CK, NO3--N content decreased 33.1%-43.8% and the rate of N2O release decreased 8-26 times under different fertilizer treatments during incubation of 0-7 days. On the contrary, NH4+-N in T28 with or without sea buckthorn pomace treatments increased by 56.5-118.8% during incubation of 7-14 days. The results indicated that this biofertilizer reduced the environmental risk associated with the accumulation of NO3--N in paddy soil and the release of N2O to the atmosphere and maintained the soil available N supply capacity. Besides, applying Bacillus T28 with sea buckthorn pomace increased the abundance of soil N functional genes such as nifH, narG, nirS, nirK, and nosZ. The 13C-PLFAs results demonstrated that this biofertilizer improves soil microbial community diversity, nutrient turnover rate and ecosystem stability by altering soil pH and total carbon (TC). In conclusion, Bacillus sp. T28 combined with sea buckthorn pomace regulated the indigenous soil microbial community structure and mitigated the environmental risk of conventional N fertilization in agroecosystems.
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Azotobacter , Bacillus , Hippophae , Microbiota , Suelo/química , Fertilizantes , Microbiología del Suelo , Bacterias , NitrógenoRESUMEN
Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.
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COVID-19 , Humanos , SARS-CoV-2 , Reacción en Cadena de la Polimerasa/métodos , Prueba de COVID-19RESUMEN
The pandemic of COVID-19 (SARS-CoV-2 disease) has been causing unprecedented health and economic impacts, alerting the world to the importance of basic sanitation and existing social inequalities. The risk of the spread and appearance of new diseases highlights the need for the removal of these pathogens through efficient techniques and materials. This study aimed to develop a polyurethane (PU) biofoam filled with dregs waste (leftover from the pulp and paper industry) for removal SARS-CoV-2 from the water. The biofoam was prepared by the free expansion method with the incorporation of 5wt% of dregs as a filler. For the removal assays, the all materials and its isolated phases were incubated for 24 h with an inactivated SARS-CoV-2 viral suspension. Then, the RNA was extracted and the viral load was quantified using the quantitative reverse transcription (RT-qPCR) technique. The biofoam (polyurethane/dregs) reached a great removal percentage of 91.55%, whereas the isolated dregs waste was 99.03%, commercial activated carbon was 99.64%, commercial activated carbon/polyurethane was 99.30%, and neat PU foam reached was 99.96% for this same property and without statistical difference. Those new materials endowed with low cost and high removal efficiency of SARS-CoV-2 as alternatives to conventional adsorbents.
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COVID-19 , SARS-CoV-2 , Humanos , Poliuretanos , Carbón Orgánico , Sensibilidad y Especificidad , ARN Viral/genéticaRESUMEN
@#Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses. Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared. The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin. Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses. Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2 × 10~2copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2 × 10~1copies/μL. Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37. 58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements. The coincidence rate of blind sample detection results by 4 laboratories was 100%. All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses. Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.
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Twenty samples of minced chicken meat procured from butcher's shops in León (Spain; 10 samples) and Vila Real (Portugal; 10 samples) were analyzed. Microbial concentrations (log10 cfu/g) of 7.53 ± 1.02 (viable aerobic microbiota), 7.13 ± 1.07 (psychrotrophic microorganisms), and 4.23 ± 0.88 (enterobacteria) were found. The detection method described in the UNE-EN ISO 11290-1 standard (based on isolation from the chromogenic medium OCLA) with confirmation by the polymerase chain reaction (PCR; lmo1030) (OCLA−PCR), revealed Listeria monocytogenes in 14 samples (70.0% of the total), nine of Spanish origin and five of Portuguese (p > 0.05). The levels of viable and inactivated L. monocytogenes in the samples were determined with a q-PCR using propidium monoazide (PMAxx) as a viability marker. Seven samples tested positive both with the OCLA−PCR and with the q-PCR, with estimated concentrations of viable cells varying between 2.15 log10 cfu/g (detection limit) and 2.94 log10 cfu/g. Three samples tested negative both with the OCLA−PCR and with the q-PCR. Seven samples were positive with the OCLA−PCR, but negative with the q-PCR, and three samples tested negative with the OCLA−PCR and positive with the q-PCR. The percentage of viable cells relative to the total ranged between 2.4% and 86.0%. Seventy isolates of L. monocytogenes (five from each positive sample) were classified in PCR serogroups with a multiplex PCR assay. L. monocytogenes isolates belonged to serogroups IIa (52 isolates; 74.3%), IIc (7; 10.0%), IVa (2; 2.9%), and IVb (9; 12.9%). The susceptibility of the 70 isolates to 15 antibiotics of clinical interest was tested. The strains presented resistance to between three and eight antibiotics. The average number of resistances was greater (p < 0.001) among strains isolated from Spanish samples (6.20 ± 1.08), than in those from Portugal (5.00 ± 1.08). In both groups of strains, a prevalence of resistance higher than 95% was observed for oxacillin, cefoxitin, cefotaxime, and cefepime. The need to handle minced chicken meat correctly, taking care to cook it sufficiently and to avoid cross-contamination, so as to reduce the danger of listeriosis, is emphasized. A combination of culture-dependent and culture-independent methods offers complementary routes for the detection in food of the cells of L. monocytogenes in various different physiological states.